High affinity TGFbeta nucleic acid ligands and inhibitors

ABSTRACT

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to TGFβ2. Included in the invention are specific RNA ligands to TGFβ2 identified by the SELEX method. Also included are RNA ligands that inhibit the interaction of TGFβ2 with its receptor.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/363,939, filed Jul. 29, 1999, entitled “High Affinity TGFβNucleic Acid Ligands and Inhibitors,” which is a continuation-in-part ofU.S. Pat. No. 6,124,449, filed Mar. 23, 1998, entitled “High AffinityTGFβ Nucleic Acid Ligands and Inhibitors,” which is acontinuation-in-part of U.S. Pat. No. 5,731,424, filed Jun. 2, 1995,entitled “High Affinity TGFβ Nucleic Acid Ligands and Inhibitors,”,which is a continuation-in-part of U.S. Pat. No. 5,475,096, filed Jun.10, 1991, entitled “Nucleic Acid Ligands,” U.S. Pat. No. 5,270,163,filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic AcidLigands,” U.S. Pat. No. 5,496,938, filed Oct. 21, 1992, entitled“Nucleic Acid Ligands to HIV-RT and HIV- 1 Rev” and U.S. patentapplication Ser. No. 08/117,991, filed Sep. 8, 1993, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” nowabandoned. U.S. Pat. No. 5,475,096 is a continuation-in-part of U.S.patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled“Systematic Evolution of Ligands by EXponential Enrichment,” nowabandoned. This application is also a continuation-in-part of U.S. Pat.No. 6,011,020, entitled “Nucleic Acid Ligand Complexes.”

FIELD OF THE INVENTION

[0002] Described herein are methods for identifying and preparing highaffinity nucleic acid ligands that bind human transforming growth factorβ2 (TGFβ2). The method utilized herein for identifying such nucleic acidligands is called SELEX, an acronym for Systematic Evolution of Ligandsby EXponential Enrichment. This invention includes high affinity nucleicacids of human TGFβ2. Further disclosed are RNA ligands to TGFβ2. Alsoincluded are oligonucleotides containing nucleotide derivatives modifiedat the 2′ position of the pyrimidines. Additionally disclosed areligands to TGFβ2 containing 2′-OCH₃ purine modifications that may havehigher stability in serum and in animals. This invention also includeshigh affinity nucleic acid inhibitors of TGFβ2. The oligonucleotideligands of the present invention are useful in any process in whichbinding to TGFβ2 is required. This includes, but is not limited to,their use as pharmaceuticals, diagnostics, imaging agents, andimmunohistochemical reagents.

BACKGROUND OF THE INVENTION

[0003] Transforming growth factors betas (TGFβs) are part of asuperfamily of proteins that includes inhibins, activins, bonemorphogenetic and osteogenic proteins, growth/differentiation factors,Mullerian-inhibiting substance, decapentaplegic and 60A (Drosophila),daf-7 and unc-129 (C. elegans), and vg1 (Xenopus) (Schlunegger andGrutter (1992) Nature 358:430-434). Three TGFβ isotypes exist in mammalsthat are called TGFβ1, TGFβ2, and TGFβ3. There is about 80% sequenceidentity between any pair of mammalian TGFβs. TGFβs bind to at least 5receptors, but only 2 or 3 of them (types I, II, and possibly V) aresignaling receptors. The intracellular signaling pathways activated byTGFβs involve SMAD proteins and are being intensively studied (Padgettet al. (1998) Pharmacol Ther 78:47-52). The signaling receptors arefound on a variety of cells. In turn, a variety of cells express TGFβs.

[0004] TGFβs are synthesized as precursors composed oflatency-associated protein (LAP) at the amino terminus and mature TGFβat the carboxyl terminus. The precursor is cleaved and assembles as ahomodimer. TGFβs are secreted from cells bound to LAP and latent TGFβbinding proteins (LTBPs). Latent TGFβs are released from LAP and LTBPand become active by a relatively uncharacterized mechanism that mayinvolve proteolysis by plasmin or regulation by thrombospondin (Crawfordet al. (1998) Cell 93:1159-70). The mature, released TGFβ homodimer hasa combined molecular weight of ˜25000 daltons (112 amino acids permonomer). TGFβ1 and TGFβ2 bind heparin and there are indications thatbasic amino acids at position 26 are required for heparin binding (Lyonet al. (1997) Jour. Biol. Chem. 272:18000-18006).

[0005] The structure of TGFβ2 has been determined using x-raycrystallography (Daopin et al. (1992) Science 257:369-373; Schluneggerand Grutter (1992) Nature 358:430-434) and is very similar to thestructure of TGFβ1. TGFβs belong to a structural family of proteinscalled the “cysteine knot” proteins that includes vascular endothelialgrowth factor, nerve growth factor, human chorionic gonadotropin, andplatelet-derived growth factor. These proteins are structurallyhomologous, but have only 10-25% primary sequence homology.

[0006] The biological activities of the TGFβs vary (Moses (1990) GrowthFactors from Genes to Clinical Application 141-155; Wahl (1994) J. Exp.Med. 180:1587-1590). In some cases they inhibit cell proliferation(Robinson et al. (1991) Cancer Res. 113:6269-6274) and in other casesthey stimulate it (Fynan and Reiss (1993) Crit. Rev. Oncogenesis4:493-540). They regulate extracellular matrix formation and remodeling(Koli and Arteaga (1996) J. Mammary Gland Bio. and Neoplasia 1:373-380).They are also are very potent immunosuppressants (Letterio and Roberts(1998) Ann. Rev. Immunol. 16:137-161). TGFβs are thought to play asignificant role in fibrotic diseases, preventing the immune system fromrejecting tumors (Fakhrai et al. (1996) Proc. Natl. Acad. USA93:2090-2914), cancer cell growth (Koli and Arteaga (1996) J. MammaryGland Bio. and Neoplasia 1:373-380; Reiss and Barcellos-Hoff (1997)Breast Cancer Res. and Treatment 45:81-85; Jennings and Pietenpol (1998)J. Neurooncol. 36:123-140), and tumor metastasis. They may haveancillary roles in autoimmune and infectious diseases. Inhibition ofTGFβ2 by an expressed antisense RNA (Fakhrai et al. (1996) Proc. Natl.Acad. USA 93:2090-2914) and by exogenous antisense oligonucleotides(Marzo et al. (1997) Cancer Research 57:3200-3207) has been reported toprevent glioma formation in rats.

[0007] The gene for mouse TGFβ2 has been deleted (Sanford et al. (1997)Development 124:2659-2670). Mice lacking TGFβ2 function die near birthand have aberrant epithelial-mesencymal interactions that lead todevelopmental defects in the heart, eye, ear, lung, limb, craniofacialarea, spinal cord, and urogenital tracts. These defects, for the mostpart, do not overlap abnormalities that have been observed in TGFβ1 andTGFβ3 knockout mice. TGFβs have also been overexpressed in cell lines ortransgeneic mice (Koli and Arteaga (1996) J. Mammary Gland Bio. andNeoplasia 1:373-380; Bottinger et al. 1997 Kidney Int. 51:1355-1360;Bottinger and Kopp (1998) Miner Electrolyte Metab 24:154-160) with avariety of effects.

[0008] A method for the in vitro evolution of nucleic acid moleculeswith high affinity binding to target molecules has been developed. Thismethod, Systematic Evolution of Ligands by EXponential enrichment,termed SELEX, is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by Exponential Enrichment,” now abandoned, U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands,” now U.S. Pat. No. 5,475,096 and U.S. patent applicationSer. No. 07/931,473, filed Aug. 17, 1992, entitled “Methods forIdentifying Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see alsoWO91/19813), each of which is herein specifically incorporated byreference. Each of these applications, collectively referred to hereinas the SELEX Patent Applications, describe a fundamentally novel methodfor making a nucleic acid ligand to any desired target molecule.

[0009] The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection theme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound to target molecules, dissociating the nucleicacid-target complexes, amplifying the nucleic acids dissociated from thenucleic acid-target complexes to yield a ligand-enriched mixture ofnucleic acids, then reiterating the steps of binding, partitioning,dissociating and amplifying through as many cycles as desired to yieldhigh affinity nucleic acid ligands to the target molecule.

[0010] The basic SELEX method may be modified to achieve specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” now abandoned, describes the use of SELEX inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA (See U.S.Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filedSep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” nowabandoned, describes a SELEX based method for selecting nucleic acidligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled“High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now abandoned, describes a method foridentifying highly specific nucleic acid ligands able to discriminatebetween closely related molecules, termed “Counter-SELEX” (See U.S. Pat.No. 5,580,737). U.S. patent application Ser. No. 08/143,564, filed Oct.25, 1993, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Solution SELEX,” now abandoned, (See also U.S. Pat. No.5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan.31, 1997, entitled “Flow Cell SELEX,” now U.S. Pat. No. 5,861,254,describe SELEX-based methods which achieve highly efficient partitioningbetween oligonucleotides having high and low affinity for a Targetmolecule. U.S. patent application Ser. No. 07/964,624, filed Oct, 21,1992, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” now U.S.Pat. No. 5,496,938, describes methods for obtaining improved NucleicAcid Ligands after the SELEX process has been performed. U.S. patentapplication Ser. No. 08/400,440, filed Mar. 8, 1995, entitled“Systematic Evolution of Ligands by EXponential Enrichment:Chemi-SELEX,” now U.S. Pat. No. 5,705,337,describes methods forcovalently linking a ligand to its target.

[0011] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability ordelivery. Examples of such modifications include chemical substitutionsat the ribose and/or phosphate and/or base positions. SpecificSELEX-identified nucleic acid ligands containing modified nucleotidesare described in U.S. patent application Ser. No. 08/117,991, filed Sep.8, 1993, entitled “High Affinity Nucleic Acid Ligands ContainingModified Nucleotides,” now abandoned, that describes oligonucleotidescontaining nucleotide derivatives chemically modified at the 5- and2′-positions of pyrimidines, as well as specific RNA ligands to thrombincontaining 2′-amino modifications (See U.S. Pat. No. 5,660,985). U.S.patent application Ser. No. 08/134,028, supra, describes highly specificnucleic acid ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-O Me). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′ Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” now abandoned, describesoligonucleotides containing various 2′-modified pyrimidines.PCT/US98/00589 (WO 98/18480), filed Jan. 7, 1998, entitled“Bioconjugation of Oligonucleotides” describes a method for identifyingbioconjugates to a target comprising nucleic acid ligands derivatizedwith a molecular entity exclusively at the 5′-position of the nucleicacid ligands.

[0012] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. patent application Ser. No. 08/284,063, filedAug. 2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459 and U.S. patentapplication Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules. Thefull text of the above described Patent applications, including but notlimited to, all definitions and descriptions of the SELEX process, arespecifically incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0013] The present invention includes methods of identifying andproducing nucleic acid ligands to transforming growth factor beta(TGFβ2) and the nucleic acid ligands so identified and produced. Inparticular, RNA sequences are provided that are capable of bindingspecifically to TGFβ2. Also included are oligonucleotides containingnucleotide derivatives modified at the 2′ position of the pyrimidines.Specifically included in the invention are the RNA ligand sequencesshown in Tables 5, 7, 8, 11, 13, 14, 16-19 and FIG. 9 (SEQ ID NOS:21-121 and 128-193). Also included in this invention are RNA ligands ofTGFβ2 that inhibit the function of TGFβ2. Also described herein are2′-OMe-modified nucleic acid ligands of TGFβ1, shown in Table 22 (SEQ IDNOS: 194-216).

[0014] Further included in this invention is a method of identifyingnucleic acid ligands and nucleic acid ligand sequences to TGFβ2,comprising the steps of (a) preparing a candidate mixture of nucleicacids, (b) contacting the candidate mixture of nucleic acids with TGFβ2,(c) partitioning between members of said candidate mixture on the basisof affinity to TGFβ2, and (d) amplifying the selected molecules to yielda mixture of nucleic acids enriched for nucleic acid sequences with arelatively higher affinity for binding to TGFβ2.

[0015] More specifically, the present invention includes the RNA ligandsto TGFβ2, identified according to the above-described method, includingthose ligands shown in Tables 5, 7, 8, 11, 13, 14, 16-19 and FIG. 9 (SEQID NOS: 21-121 and 128-193). Also included are nucleic acid ligands toTGFβ2 that are substantially homologous to any of the given ligands andthat have substantially the same ability to bind TGFβ2 and inhibit thefunction of TGFβ2. Further included in this invention are nucleic acidligands to TGFβ2 that have substantially the same structural form as theligands presented herein and that have substantially the same ability tobind TGFβ2 and inhibit the function of TGFβ2.

[0016] The present invention also includes other modified nucleotidesequences based on the nucleic acid ligands identified herein andmixtures of the same.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 shows a flow chart summarizing the various SELEXexperiments done with TGFβ2. The length of the arrowheads corresponds tothe round number shown to the left. Connected arrowheads indicatebranches in the SELEX experiments where a pool was used to start a newbranch. Under each arrowhead the fold improvement in affinity is alsoshown.

[0018]FIG. 2 shows activity of TGFβ2 following amine coupling on aBIAcore carboxymethylcellulose (CM5) chip. A CM5 chip was loaded withTGFβ2 using NHS-EDC coupling as described in the Materials and Methodsat about 18, 718, and 1692 response units for flow cell (FC) 1, 2, and3, respectively. FC-4 was left blank as a control and was used tonormalize the signals from the other FCs. The chip was then exposed to10 nM of either (FIG. 2A) latency associated peptide (LAP) or (FIG. 2B)TGFβ soluble receptor III (sRIII) at 20 μl/min in binding buffer. Datawere collected for an association and a dissociation phase as shown. Thesignal from FC-4 was subtracted from the other FCs.

[0019]FIG. 3 shows affinity improvement during the spr SELEX. A CM5 chipwas loaded with TGFβ2 using NHS-EDC coupling as described in theMaterials and Methods at about 18, 718, and 1692 response units for flowcell (FC) 1, 2, and 3, respectively. FC-4 was left blank as a controland was used to normalize the signals from the other FCs. The chip wasthen exposed to 1 μM of RNA pools from the SELEX rounds (Rd) as shown at20 μl/min in binding buffer. Data were collected for an association anda dissociation phase as shown. The signal from FC-4 was subtracted fromthe other FCs.

[0020]FIG. 4 shows nitrocellulose filter binding curves with pools fromthe spr SELEX. High specific activity internally labeled RNA was usedfrom rounds (R) as shown. Labeled RNA was incubated with variousconcentrations of TGFβ2 in the presence of 100,000 fold molar excessunlabelled tRNA. Bound RNA was partitioned by nitrocellulose filtrationand quantitated. Data were analyzed as described in the Materials andMethods.

[0021]FIG. 5 shows nitrocellulose filter binding curves with variouspools. High specific activity internally labeled RNA was used fromrounds (R) as shown. Labeled RNA was incubated with variousconcentrations of TGFβ2 (no competitor tRNA was used). Bound RNA waspartitioned by nitrocellulose filtration and quantitated. Data wereanalyzed as described in the Materials and Methods.

[0022]FIG. 6 shows specificity of the bioactivity of lead TGFβ1 andTGFβ2 aptamers and comparison with commercial antibody preparations. RNAwas either synthesized by phosphoramidite chemistry (NX22283) or by invitro transcription. Indicator cells (mink lung epithelial cells) wereincubated with either TGFβ1, TGFβ2 or TGFβ3 and dilutions of RNA orantibody as described. The extent of cell proliferation was measured by³H-thymidine incorporation and the data were analyzed as described. Thepoints represent an average of n=2−6 and error bars are standard errors.Symbols designated by TGFβ1, TGFβ2 or TGFβ3 indicate data obtained fromcells treated with either TGFβ1, TGFβ2 or TGFβ3, respectively. MAB andpAB designate monoclonal and polyclonal antibodies, respectively.Random, NX22283 and 40-03 designate the use of random RNA, the TGFβ2 orthe TGFβ1 lead aptamer, respectively. The aptamer 40-03 was described inthe TGFβ1 patent (U.S. Pat. No. 6,124,449, entitled “High-Affinity TGFβNucleic Acid Ligands and Inhibitors”).

[0023]FIG. 7 shows boundaries of TGFβ2 ligands 14-1, 21-21 and 21-4. RNAaptamers were end labeled at the 5′ end (3′B) or at the 3′ end (5′B),partially hydrolyzed at high pH, and partitioned for binding to TGFβ2 bynitrocellulose filtration as described in the Materials and Methods. Theamounts of TGFβ2 used for binding partitioning is as shown. RecoveredRNA was analyzed on high resolution sequencing gels and visualized byautoradiography. Unselected hydrolyzed RNA was used as a marker (Alk.hydr.) to align the banding pattern to the sequence of each ligand. Theobserved boundary bands are shown with (*) and their position in thesequence pattern is shown by arrowheads. No protein and input lanes showthe background binding to nitrocellulose and the starting unhydrolyzedRNA. The observed boundaries for each ligand is summarized at the bottomof the figure.

[0024]FIG. 8 shows the putative structures of TGFβ2 aptamers. Theminimal required sequences were fit into similar structures. Ligand14i-1t5-41 and 21a-4(ML-110) were transcribed in vitro and containedextra bases at their 5′ ends (shown in lower case) to allow efficient invitro transcription. Bold-faced letters indicate positions that areidentical to invariant positions of the biased SELEX with the 21-21sequence.

[0025]FIG. 9 shows the molecular description of NX22323 40 k PEG. rG2′-OH G; rA=2′-OH A; FU=2′-FU; FC=2′-FC.

[0026]FIG. 10 shows the putative structure of lead truncate ligand CD70.Lower case letters indicate positions requiring 2′-OH.  indicates GUbase pairing.

[0027]FIG. 11 shows the pharmacokinetics of TGFβ aptamer in spraguedawley rats.

DETAILED DESCRIPTION OF THE INVENTION

[0028] This application describes high-affinity nucleic acid ligands toTGFβ2 identified through the method known as SELEX. SELEX is describedin U.S. patent application Ser. No. 07/536,428, entitled “SystematicEvolution of Ligands by EXponential Enrichment,” now abandoned, U.S.patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled“Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096 and U.S. patentapplication Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Methodsfor Identifying Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (seealso WO91/19813). These applications, each specifically incorporatedherein by reference, are collectively called the SELEX PatentApplications. Nucleic Acid Ligands to TGFβ have been identified throughthe SELEX method. These TGFβNucleic Acid Ligands are described in U.S.patent application Ser. No. 08/458,423, filed Jun. 2, 1995, entitled,“High Affinity TGFβ Nucleic Acid Ligands and Inhibitors,” now U.S. Pat.No. 5,731,144 and U.S. patent application Ser. No. 09/046,247, filedMar. 23, 1998, entitled “High Affinity TGFβ Nucleic Acid Ligands andInhibitors,” and U.S. patent application Ser. No. 09/275,850, filed Mar.24, 1999, entitled “Truncation SELEX Method.” These applications arespecifically incorporated herein in their entirety.

[0029] Certain terms used to described the invention herein are definedas follows:

[0030] “Nucleic Acid Ligand” as used herein is a non-naturally occurringnucleic acid having a desirable action on a target. Nucleic Acid Ligandsare also referred to herein as “aptarners. ” A desirable actionincludes, but is not limited to, binding of the target, catalyticallychanging the target, reacting with the target in a way whichmodifies/alters the target or the functional activity of the target,covalently attaching to the target as in a suicide inhibitor, andfacilitating the reaction between the target and another molecule. Inthe preferred embodiment, the desirable action is specific binding to atarget molecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the nucleic acid ligand isnot a nucleic acid having the known physiological function of beingbound by the target molecule. Nucleic acid ligands include nucleic acidsthat are identified from a candidate mixture of nucleic acids, saidnucleic acid ligand being a ligand of a given target by the methodcomprising: a) contacting the candidate mixture with the target, whereinnucleic acids having an increased affinity to the target relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; b) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; and c) amplifying the increasedaffinity nucleic acids to yield a ligand-enriched mixture of nucleicacids. “Candidate Mixture” is a mixture of nucleic acids of differingsequence from which to select a desired ligand. The source of acandidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process. “Nucleic Acid” means either DNA, RNA,single-stranded or double-stranded and any chemical modificationsthereof. Modifications include, but are not limited to, those whichprovide other chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil,backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

[0031] “SELEX” methodology involves the combination of selection ofnucleic acid ligands which interact with a target in a desirable manner,for example binding to a protein, with amplification of those selectednucleic acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain nucleic acidligands to TGFβ2. The SELEX methodology is described in the SELEX PatentApplications.

[0032] “Target” means any compound or molecule of interest for which aligand is desired. A target can be a protein, peptide, carbohydrate,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, substrate, metabolite, transition state analog, cofactor,inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.In this application, the target is TGFβ2.

[0033] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0034] 1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0035] 2) The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

[0036] 3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

[0037] 4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0038] 5) By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer weakly bindingsequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0039] The SELEX Patent Applications describe and elaborate on thisprocess in great detail. Included are targets that can be used in theprocess; methods for partitioning nucleic acids within a candidatemixture; and methods for amplifying partitioned nucleic acids togenerate enriched candidate mixture. The SELEX Patent Applications alsodescribe ligands obtained to a number of target species, including bothprotein targets where the protein is and is not a nucleic acid bindingprotein.

[0040] The SELEX method further encompasses combining selected nucleicacid ligands with lipophilic or non-immunogenic, high molecular weightcompounds in a diagnostic or therapeutic complex as described in U.S.patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes,” now U.S. Pat. No. 6,011,020. VEGFnucleic acid ligands that are associated with a Lipophilic Compound,such as diacyl glycerol or dialkyl glycerol, in a diagnostic ortherapeutic complex are described in U.S. patent application Ser. No.08/739,109, filed Oct. 25, 1996, entitled “Vascular Endothelial GrowthFactor (VEGF) Nucleic Acid Ligand Complexes,” now U.S. Pat. No.5,859,228. VEGF nucleic acid ligands that are associated with aLipophilic Compound, such as a glycerol lipid, or a non-immunogenic,high molecular weight Compound, such as polyalkylene glycol, are furtherdescribed in U.S. patent application Ser. No. 08/897,351, filed Jul. 21,1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes,” now U.S. Pat. No. 6,051,698. VEGF nucleic acidligands that are associated with a non-immunogenic, high molecularweight compound or lipophilic compound are also further described inPCT/US 97/18944 (WO98/18480), filed Oct. 17, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes”. Each ofthe above described Patent applications which describe modifications ofthe basic SELEX procedure are specifically incorporated by referenceherein in their entirety.

[0041] In certain embodiments of the present invention it is desirableto provide a complex comprising one or more nucleic acid ligands toTGFβ2 covalently linked with a non-immunogenic, high molecular weightcompound or lipophilic compound. A complex as used herein describes themolecular entity formed by the covalent linking of the nucleic acidligand of TGFβ2 to a non-immunogenic, high molecular weight compound. Anon-immunogenic, high molecular weight compound is a compound betweenapproximately 100 Da to 1,000,000 Da, more preferably approximately 1000Da to 500,000 Da, and most preferably approximately 1000 Da to 200,000Da, that typically does not generate an immunogenic response. For thepurposes of this invention, an immunogenic response is one that causesthe organism to make antibody proteins. In one preferred embodiment ofthe invention, the non-immunogenic, high molecular weight compound is apolyalkylene glycol. In the most preferred embodiment, the polyalkyleneglycol is polyethylene glycol (PEG). More preferably, the PEG has amolecular weight of about 10-80K. Most preferably, the PEG has amolecular weight of about 20-45K. In certain embodiments of theinvention, the non-immunogenic, high molecular weight compound can alsobe a nucleic acid ligand.

[0042] In another embodiment of the invention it is desirable to have acomplex comprised of a nucleic acid ligand to TGFβ2 and a lipophiliccompound. Lipophilic compounds are compounds that have the propensity toassociate with or partition into lipid and/or other materials or phaseswith low dielectric constants, including structures that are comprisedsubstantially of lipophilic components. Lipophilic compounds includelipids as well as non-lipid containing compounds that have thepropensity to associate with lipid (and/or other materials or phaseswith low dielectric constants). Cholesterol, phospholipid, and glycerollipids, such as dialkylglycerol, diacylglycerol, and glycerol amidelipids are further examples of lipophilic compounds. In a preferredembodiment, the lipophilic compound is a glycerol lipid.

[0043] The non-immunogenic, high molecular weight compound or lipophiliccompound may be covalently bound to a variety of positions on thenucleic acid ligand to TGFβ2, such as to an exocyclic amino group on thebase, the 5-position of a pyrimidine nucleotide, the 8-position of apurine nucleotide, the hydroxyl group of the phosphate, or a hydroxylgroup or other group at the 5′ or 3′ terminus of the nucleic acid ligandto TGFβ2. In embodiments where the lipophilic compound is a glycerollipid, or the non-immunogenic, high molecular weight compound ispolyalkylene glycol or polyethylene glycol, preferably thenon-immunogenic, high molecular weight compound is bonded to the 5′ or3′ hydroxyl of the phosphate group thereof. In the most preferredembodiment, the lipophilic compound or non-immunogenic, high molecularweight compound is bonded to the 5′ hydroxyl of the phosphate group ofthe nucleic acid ligand. Attachment of the non-immunogenic, highmolecular weight compound or lipophilic compound to the nucleic acidligand of TGFβ can be done directly or with the utilization of linkersor spacers.

[0044] A “linker” is a molecular entity that connects two or moremolecular entities through covalent bonds or non-covalent interactions,and can allow spatial separation of the molecular entities in a mannerthat preserves the functional properties of one or more of the molecularentities. A linker can also be known as a “spacer.”

[0045] The complex comprising a nucleic acid ligand to TGFβ2 and anon-immunogenic, high molecular weight compound or lipophilic compoundcan be further associated with a lipid construct. Lipid constructs arestructures containing lipids, phospholipids, or derivatives thereofcomprising a variety of different structural arrangements which lipidsare known to adopt in aqueous suspension. These structures include, butare not limited to, lipid bilayer vesicles, micelles, liposomes,emulsions, lipid ribbons or sheets, and may be complexed with a varietyof drugs and components which are known to be pharmaceuticallyacceptable. In the preferred embodiment, the lipid construct is aliposome. The preferred liposome is unilamellar and has a relative sizeless than 200 nm. Common additional components in lipid constructsinclude cholesterol and alpha-tocopherol, among others. The lipidconstructs may be used alone or in any combination which one skilled inthe art would appreciate to provide the characteristics desired for aparticular application. In addition, the technical aspects of lipidconstructs and liposome formation are well known in the art and any ofthe methods commonly practiced in the field may be used for the presentinvention.

[0046] The SELEX method further comprises identifying bioconjugates to atarget. Copending PCT Patent Application No. PCT/US 98/00589 (WO98/18480), filed Jan. 7, 1998, entitled “Bioconjugation ofOligonucleotides” describes a method for enzymatically synthesizingbioconjugates comprising RNA derivatized exclusively at the 5′-positionwith a molecular entity, and a method for identifying bioconjugates to atarget comprising nucleic acid ligands derivatized with a molecularentity exclusively at the 5′-position of the nucleic acid ligands. Abioconjugate as used herein refers to any oligonucleotide which has beenderivatized with another molecular entity. In the preferred embodiment,the molecular entity is a macromolecule. As used herein, a macromoleculerefers to a large organic molecule. Examples of macromolecules include,but are not limited to nucleic acids, oligonucleotides, proteins,peptides, carbohydrates, polysaccharides, glycoproteins, lipophiliccompounds, such as cholesterol, phospholipids, diacyl glycerols anddialkyl glycerols, hormones, drugs, non-immunogenic high molecularweight compounds, fluorescent, chemiluminescent and bioluminescentmarker compounds, antibodies and biotin, etc. without limitation. Incertain embodiments, the molecular entity may provide certain desirablecharacteristics to the nucleic acid ligand, such as increasing RNAhydrophobicity and enhancing binding, membrane partitioning and/orpermeability. Additionally, reporter molecules, such as biotin,fluorescein or peptidyl metal chelates for incorporation of diagnosticradionuclides may be added, thus providing a bioconjugate which may beused as a diagnostic agent.

[0047] The methods described herein and the nucleic acid ligandsidentified by such methods are useful for both therapeutic anddiagnostic purposes. Therapeutic uses include the treatment orprevention of diseases or medical conditions in human patients.Therapeutic uses may also include veterinary applications.

[0048] Diagnostic utilization may include both in vivo or in vitrodiagnostic applications. The SELEX method generally, and the specificadaptations of the SELEX method taught and claimed herein specifically,are particularly suited for diagnostic applications. SELEX identifiesnucleic acid ligands that are able to bind targets with high affinityand with surprising specificity. These characteristics are, of course,the desired properties one skilled in the art would seek in a diagnosticligand.

[0049] The nucleic acid ligands of the present invention may beroutinely adapted for diagnostic purposes according to any number oftechniques employed by those skilled in the art or by the methodsdescribed in PCT/US 98/00589 (WO 98/18480). Diagnostic agents need onlybe able to allow the user to identify the presence of a given target ata particular locale or concentration. Simply the ability to form bindingpairs with the target may be sufficient to trigger a positive signal fordiagnostic purposes. Those skilled in the art would also be able toadapt any nucleic acid ligand by procedures known in the art toincorporate a labeling tag in order to track the presence of suchligand. Such a tag could be used in a number of diagnostic procedures.The nucleic acid ligands to TGFβ2 described herein may specifically beused for identification of the TGFβ2 protein.

[0050] SELEX provides high affinity ligands of a target molecule. Thisrepresents a singular achievement that is unprecedented in the field ofnucleic acids research. The present invention applies the SELEXprocedure to the specific target of TGFβ1. In the Example section below,the experimental parameters used to isolate and identify the nucleicacid ligands to TGFβ2 are described.

[0051] In order to produce nucleic acids desirable for use as apharmaceutical, it is preferred that the nucleic acid ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the nucleic acid ligandhave the highest possible affinity to the target.

[0052] In co-pending and commonly assigned U.S. patent application Ser.No. 07/964,624, filed Oct. 21, 1992 ('624), methods are described forobtaining improved nucleic acid ligands after SELEX has been performed.The '624 application, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1Rev,” now issued as U.S. Pat. No. 5,496,938, is specificallyincorporated herein by reference.

[0053] In the present invention, SELEX experiments were performed inorder to identify RNA ligands with specific high affinity for TGFβ2 fromdegenerate libraries containing 33, 34 or 40 random positions (33N, 34Nor 40N) (Table 1). This invention includes the specific RNA ligands toTGFβ2 shown in Tables 5, 7, 8, 11, 13, 14, 16-19 and FIG. 9 (SEQ ID NOS:21-121 and 128-193), identified by the methods described in Example 1.This invention further includes RNA ligands to TGFβ2 which inhibit TGFβ2function, presumably by inhibiting the interaction of TGFβ2 with itsreceptor. The scope of the ligands covered by this invention extends toall nucleic acid ligands of TGFβ2, modified and unmodified, identifiedaccording to the SELEX procedure. More specifically, this inventionincludes nucleic acid sequences that are substantially homologous to theligands shown in Tables 5, 7, 8, 11, 13, 14, 16-19 and FIG. 9 (SEQ IDNOS: 21-121 and 128-193). By substantially homologous it is meant adegree of primary sequence homology in excess of 70%, most preferably inexcess of 80%, and even more preferably in excess of 90%, 95% or 99%.The percentage of homology as described herein is calculated as thepercentage of nucleotides found in the smaller of the two sequenceswhich align with identical nucleotide residues in the sequence beingcompared when 1 gap in a length of 10 nucleotides may be introduced toassist in that alignment. A review of the sequence homologies of theligands of TGFβ2, shown in Tables 5, 7, 8, 11, 13, 14, 16-19 and FIG. 9(SEQ ID NOS: 21-121 and 128-193) shows that some sequences with littleor no primary homology may have substantially the same ability to bindTGFβ2. For these reasons, this invention also includes nucleic acidligands that have substantially the same structure and ability to bindTGFβ2 as the nucleic acid ligands shown in Tables 5, 7, 8, 11, 13, 14,16-19 and FIG. 9 (SEQ ID NOS: 21-121 and 128-193). Substantially thesame ability to bind TGFβ2 means that the affinity is within one or twoorders of magnitude of the affinity of the ligands described herein. Itis well within the skill of those of ordinary skill in the art todetermine whether a given sequence ——substantially homologous to thosespecifically described herein——has substantially the same ability tobind TGFβ.

[0054] This invention also includes nucleic acid ligands that havesubstantially the same postulated structure or structural motifs.Substantially the same structure or structural motifs can be postulatedby sequence aligmnent using the Zukerfold program (see Zuker (1989)Science 244:48-52). As would be known in the art, other computerprograms can be used for predicting secondary structure and structuralmotifs. Substantially the same structure or structural motif of nucleicacid ligands in solution or as a bound structure can also be postulatedusing NMR or other techniques as would be known in the art.

[0055] One potential problem encountered in the therapeutic,prophylactic, and in vivo diagnostic use of nucleic acids is thatoligonucleotides in their phosphodiester form may be quickly degraded inbody fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the nucleic acid ligand can be made toincrease the in vivo stability of the nucleic acid ligand or to enhanceor to mediate the delivery of the nucleic acid ligand. See, e.g., U.S.patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”now abandoned and U.S. patent application Ser. No. 08/434,465, filed May4, 1995, entitled “Nucleic Acid Ligand Complexes,” now U.S. Pat. No.6,011,020, which are specifically incorporated herein by reference.Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil,backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

[0056] Where the nucleic acid ligands are derived by the SELEX method,the modifications can be pre- or post- SELEX modifications. Pre-SELEXmodifications yield nucleic acid ligands with both specificity for theirSELEX Target and improved in vivo stability. Post-SELEX modificationsmade to 2′-OH nucleic acid ligands can result in improved in vivostability without adversely affecting the binding capacity of thenucleic acid ligand. The preferred modifications of the nucleic acidligands of the subject invention are 5′ and 3′ phosphorothioate cappingand/or 3′-3′ inverted phosphodiester linkage at the 3′ end. In onepreferred embodiment, the preferred modification of the nucleic acidligand is a 3′-3′ inverted phosphodiester linkage at the 3′ end.Additional 2′-fluoro (2′-F) and/or 2′-amino (2′-NH₂) and/or 2′-O methyl(2′-OMe) and/or 2′-OCH₃ modification of some or all of the nucleotidesis preferred. Described herein are nucleic acid ligands that were 2′-Fmodified and incorporated into the SELEX process. Also described hereinare nucleic acid ligands that were 2′-OCH₃ modified after the SELEXprocess was performed.

[0057] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0058] As described above, because of their ability to selectively bindTGFβ2, the nucleic acid ligands to TGFβ2 described herein are useful aspharmaceuticals. This invention, therefore, also includes a method fortreating TGFβ2-mediated pathological conditions by administration of anucleic acid ligand capable of binding to TGFβ2.

[0059] Therapeutic compositions of the nucleic acid ligands may beadministered parenterally by injection, although other effectiveadministration forms, such as intraarticular injection, inhalant mists,orally active formulations, transdermal iontophoresis or suppositories,are also envisioned. One preferred carrier is physiological salinesolution, but it is contemplated that other pharmaceutically acceptablecarriers may also be used. In one preferred embodiment, it is envisionedthat the carrier and the ligand constitute a physiologically-compatible,slow release formulation. The primary solvent in such a carrier may beeither aqueous or non-aqueous in nature. In addition, the carrier maycontain other pharmacologically-acceptable excipients for modifying ormaintaining the pH, osmolarity, viscosity, clarity, color, sterility,stability, rate of dissolution, or odor of the formulation. Similarly,the carrier may contain still other pharmacologically-acceptableexcipients for modifying or maintaining the stability, rate ofdissolution, release, or absorption of the ligand. Such excipients arethose substances usually and customarily employed to formulate dosagesfor parental administration in either unit dose or multi-dose form.

[0060] Once the therapeutic composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or dehydrated or lyophilized powder. Such formulations may be storedeither in a ready to use form or requiring reconstitution immediatelyprior to administration. The manner of administering formulationscontaining nucleic acid ligands for systemic delivery may be viasubcutaneous, intramuscular, intravenous, intranasal or vaginal orrectal suppository.

[0061] The following Examples are provided to explain and illustrate thepresent invention and are not intended to be limiting of the invention.Example 1 describes the various materials and experimental proceduresused in Examples 2-5. Example 2 describes the isolation andcharacteristics of Nucleic Acid Ligands that bind human TGFβ2. Example 3describes the Nucleic Acid Ligands isolated by the SELEX method using abiased round 0 library, the sequences of TGFβ2 Nucleic Acid Ligandsisolated from the biased SELEX process, and the binding of Nucleic AcidLigands isolated from the biased SELEX process. Example 4 describessubstitutions of 2′-OH purines with 2′OCH₃ purines in NX22284 andNX222385. Example 5 describes the pharmacokinetic properties of NX22323. Example 6 describes 2′-OMe modification of lead TGFβ1 truncate ligandCD70.

EXAMPLES Example 1 Experimental Procedures

[0062] Materials and Methods

[0063] Monoclonal and polyclonal antibodies that recognize human TGFβ1,TGFβ2 or TGFβ3 were purchased from R&D Systems, Inc. (Minneapolis,Minn.). DNA oligonucleotides were purchased from Operon, Inc. (Alameda,Calif.) or Oligos, Etc. (Redding Center, Conn.). The BIAcore 2000 andIAsys plus instruments are products of Biacore, Inc. (Paramus, N.J.) andAffinity Sensors, Inc. (Cambridge, U. K.), respectively. Nitrocellulosefilters and filtering manifolds were obtained from Millipore (Bedford,Md.). Mink lung epithelial cells (#CCL64) were purchased from theAmerican Type Culture Collection (Rockville, Md.). The cloning vectorspCR-Script and pUC9 were obtained in-house or from Stratagene, Inc. (LaJolla, Calif.) or Life Technologies, Inc. (Gaithersburg, Md.),respectively. E coil strains were obtained from Stratagene. The QlAprepspin miniprep kit was from QIAgen, Inc. (Chatsworth, Calif.). The BigDye sequencing kit and model 377 sequencer can be purchased from AppliedBiosystems (Foster City, Calif.). T7 RNA polymerase and Thermusaquaticus DNA polymerase were purchased from Enzyco, Inc. (Denver,Colo.) and Perkin Elmer (Norwalk, Conn.), respectively. All restrictionenzymes were purchased from New England Biolabs. E. coli RNase H wasobtained from Boehringer Mannheim. All synthetic nucleic acids with aname that begins with “NX” were synthesized at NeXstar Pharmaceuticals,Inc. (Boulder, Colo.) using an ABI model 394 DNA/RNA synthesizer(Applied Biosystems). Yeast tRNA (type X-SA) and porcine intestinalmucosca-derived heparin (molecular weight 5000), were purchased fromSigma (St. Louis, Mo.) and Calbiochem (La Jolla, Calif.), respectively.

[0064] Preparation of Round 0 Nucleic Acid Library

[0065] The initial (round 0) library of ribonucleic acid molecules thatwas used to isolate TGFβ2 nucleic acid ligands was generated as follows.Two DNA oligonucleotides (40 N7 round 0 DNA template and 5′N7 primer)were annealed and filled in with Klenow to produce a 40 N7 round 0 DNAtranscription template (Table 1). This template was transcribed using T7RNA polymerase, 3 mM 2′-fluoro uridine and cytosine, 1 mM 2′-hydroxylguanosine and adenine, and α³²P-ATP as described in (Fitzwater andPolisky (1996) Meth. Enz. 267:275-301). This resulted in a round 0 40N7nucleic acid pool with the following sequence which has 5′ and 3′“fixed”regions and a 40 base long random sequence region:5′-GGGAGGACGAUGCGG-40N-CAGACGACUCGCCCGA-3′ round 0 40N7 nucleic acid(SEQ ID NO:6)  5′ fixed region  random region  3′fixed region

[0066] TABLE 1 A═2′-OH A; C═2′-F C; G═2′-OH G; U═2′-F U

[0067] Spot SELEX

[0068] Spot SELEX was performed as described in U.S. patent applicationSer. No. 08/477,527, filed Jun. 7, 1995, entitled “High Affinity NucleicAcid Ligands of Cytokines,” now U.S. Pat. No. 5,972,599, which is herebyincorporated by reference in its entirety, using nucleic acid that wasinternally labeled using α-³²P ATP. The conditions and progress of thisSELEX experiment are summarized in Table 3. Briefly, human TGFβ2 (or noprotein) was applied to a 13 mm diameter nitrocellulose filter andallowed to absorb but not completely dry. The filter was incubated withRNA in Dulbecco's phosphate-buffered saline, 1 mM MgCl₂ and then washedas summarized in Table 3. Filter-bound and protein-bound nucleic acidwas visualized and quantitated on an Instant Imager (Packard InstrumentCo., Downers Grove, IL) and the protein-bound nucleic acid was eluted in50% phenol, 4 M urea for 45 minutes at 65° C. Eluted nucleic acid wasethanol precipitated and then reverse transcribed using avianmyeloblastosis virus reverse transcriptase and subjected to thepolymerase chain reaction using 5′N7 and 3′N7 primers for 15 cycles.This resulting transcription template was transcribed with T7 RNApolymerase in the presence of 2′-fluoro pyrimidine nucleotides, 2′-OHpurine ribonucleotides, and α-³²P-ATP, and carried to the next spotround. The pool from the first spot round was also transcribed as abovein the absence of α-³²P-ATP for use in round 2 of the surface plasmonresonance biosensor SELEX.

[0069] Surface Plasmon Resonance Biosensor SELEX

[0070] Rounds 2-spr through 9-spr were done using surface plasmonresonance biosensor technology on a BIAcore model 2000 instrument. Forthis experiment 1XDPBS, 1 mM MgCl₂, 0.005% P20 surfactant(cat#BR-1000-54, Biacore, Inc., Piscataway, N.J.) was used as therunning buffer. TGFβ2 was amine coupled onto a CM5 BIACORE chip(Biacore, Inc., Piscataway, N.J.) using the Biacore amine coupling kit(cat#BR-1000-50, Biacore, Inc., Piscataway, N.J.) per manufacturer'sinstructions. Briefly, TGFβ2 aliquots (3 μl, in 4 mM HCl at 100 μg/ml)were diluted in 30 μl of 10 mM CH₃COONa, pH 5.0 and injected on anEDC-NHS activated chip at 25° C., 5 μl/min, in different volumes toachieve different loading levels, as measured in response units (RU).Following coupling, the chip was washed with 3 M NaCl for about 1.5 minat 10 μl/min. Under these experimental conditions, TGFβ2 loading of 15RU/μl could be achieved. TGFβ2 was loaded in flow cells 1, 2, and 3 ,while flow cell 4 was kept blank for control and backgroundsubtractions. Before use, the chip was tested for activity by testingbinding of LAP and or soluble receptor III (R&D Systems, Minneapolis,Minn.) at 37° C. At the end of each test injections the chip wasregenerated using 1 min wash with 10 mM NaOH. For SELEX rounds, RNApools, generated by in vitro transcription without any labelednucleotides, were in running buffer and were injected over the TGFβ2loaded CM5 chips at 5 μl/min at 37° C. The concentration and volume ofthe RNA pools used at each round are as shown in Table 4. At each roundthe RNA pools were applied in 40 μl injections and each injection cyclewas followed by a dissociation phase where the chip was washed withDPBS, 1 mM MgCl₂ at 20 μl/min while three 100 μl fractions (5 min each)were collected. Following the last injection-dissociation cycle, thechip was treated with 0.25% SDS and the eluted RNA was collected as thefinal fraction. The third fractions of each injection cycle and the SDSelution were pooled and amplified by RT/PCR to generate the templatepool for the next SELEX round.

[0071] Resonant Mirror Optical Biosensor SELEX

[0072] Rounds 10-rm through 13-rm were done using an IASYS plus resonantmirror optical biosensor instrument. Round 9-spr from the surfaceplasmon resonance SELEX was used as the starting material. For thisexperiment, 1XDPBS, 1 mM MgCl₂, 0.005% P20 surfactant (cat#BR-1000-54,Biacore, Inc., Piscataway, N.J.) were used as the running buffer. TGFβ2was amine coupled onto a CMD IASYS cuvette (Affinity Sensors, Cambridge,UK) according to the manufacturer's protocol. Briefly, the CMD cuvettewas activated with 0.2 M EDC, 0.05 M NHS for 10 minutes, and TGFβ2 wascoupled by injection 35 μl of 0.4 μM TGFβ2, 10 mM CH₃COONa, pH 5.5 in 35μl of 10 mM CH₃COONa, pH 5.5. The coupling reaction was at 25° C. forabout 10 minutes and resulted in about 2,000 Arcsec of signal. Unreactedsites were capped by exposing the cuvette in 1 M ethanolamine for 1-2minutes. Following coupling and capping the cuvette was exposed to 3 MNaCl for 1-2 minutes and was ready for use. The cuvettes were routinelytested for activity by measuring binding of LAP and or soluble receptorIII (R&D Systems, Minneapolis, Minn.) at 37° C. At the end of each testinjection the chip was regenerated using a 1 minute wash with 50 mMNaCO₃. For SELEX rounds, RNA pools, generated by in vitro transcriptionwithout any labeled nucleotides, were in running buffer. They wereinjected in the TGFβ2 loaded CMD cuvette and incubated for 27-60 minutes(Table 6) under 100% steering at 37° C. Following binding, the RNA wasreplaced with buffer and bound RNA was observed to dissociate from thecuvette surface. Dissociation was allowed for 30-150 minutes (Table 6)at 37° C. while the buffer was exchanged several times to avoidevaporation. Following dissociation, the remaining RNA was eluded withH₂0 or 0.25% SDS and the RNA was amplified as above and carried to thenext SELEX round. SELEX using filter partitioning and polyanioncompetition For rounds 9b through 22a, SELEX using filter partitioningwas performed essentially as described in (Fitzwater and Polisky (1996)Meth. Enz. 267:275-301) except that 1) heparin or yeast tRNA wasincluded to compete off ligands that bound nonspecifically, 2) thebinding buffer was HBSMCK (50 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM MgCl₂,1 mM CaCl₂, 3 mM KCl), 3) extensive efforts were undertaken to reducefilter binding sequences (preadsorbtion of nucleic acid onto filtersafter elution and transcription, blocking of filters with tRNA andbovine serum albumin prior to partitioning, addition of 0.5 M urea tothe wash buffer) and 4) the transcripts were initiated with a 5:1 molarmixture of guanosine:2′-fluoro-nucleotides. Initiation with guanosineallows nucleic acids to be used in SELEX or bioactivity assays withoutradiolabeling and alleviates a phosphatase step if the nucleic acid isto be 5′-end radiolabeled for binding studies.

[0073] Round 8-spr from the surface plasmon resonance SELEX was used asthe starting material. From rounds 9b to 14i, the SELEX process wasperformed using protein-excess conditions. The concentrations of nucleicacid and protein were equimolar in round 15c. Nucleic acid-excessconditions were used from rounds 16a to 22a (Table 2). Competitors(yeast tRNA and heparin) were used from rounds 9b to 14i. Filters werewashed with 10-15 ml HBSMCK buffer from rounds 9b to 12d and increasingamounts (5-50 ml) of HBSMCK, 0.5 M urea from rounds 13i to 22a.

[0074] Sequencing of Nucleic Acid Ligand Pools

[0075] Nucleic acid pools were sequenced as described in (Fitzwater andPolisky (1996) Meth. Enz. 267:275-301).

[0076] Screening Nucleic Acid Ligand Pools using Ligand-specific ReverseTranscription-polymerase Chain Reactions

[0077] Nucleic acids from the various pools was reverse transcribed withclone-“specific” primers (ML-85 for ligand 14i-1 and ML-81 for ligand21a-21) for 12, 15 or 18 cycles. Mixtures of pure nucleic acid ligandsand round 0 40N7 nucleic acid that contained 10%, 3%, 1%, 0.3%, or 0.1%ligand were processed in the same manner and served to quantitatesignals from RT-PCR of the nucleic acid pools.

[0078] Cloning, Screening and Sequencing of Nucleic Acid Ligands

[0079] Nucleic acid ligands were cloned using two methods. In one methodthe ligands were directly cloned into pCR-Script according to themanufacturer's instructions and transformed into E. Coli strain XL-1Blue MRF′ Kan. In the other method the double-stranded DNA transcriptiontemplate was amplified by PCR using primers ML-34 and ML-78, digestedwith BamHI and EcoRi restriction enzymes, and cloned into BamHI andEcoRI-digested pUC9. The ligation was transformed into E. coli strainDH5α. Colonies were selected on ampicillin plates and screened forinserts by PCR using vector-specific primers (RSP and FSP2). Typically90%-100% of the clones had inserts. Some colonies or nucleic acid poolswere also screened using 14i-1, 21a-4 or 21a-21 ligand-specific primers(ML-79, ML-81 and ML-85, respectively) in an attempt to identify clonesthat were different from those already isolated.

[0080] Plasmid minipreps from the transformants were prepared using theQlAprep spin miniprep kit (QIAGEN, Inc., Valencia, Calif.) or PERFECTprep plasmid DNA kit (5′3′, Inc., Boulder, Colo.). Sequencing reactionswere performed with the Big Dye kit and a sequencing primer (RSP2). Thesequencing products were analyzed on an ABI model 377 sequencer.

[0081] Nucleic Acid Ligand Boundaries

[0082] The boundaries (5′ and 3′ end) of the smallest ligand that canbind TGFβ2 was determined essentially as described in (Fitzwater andPolisky (1996) Meth. Enz. 267:275-301). The protein concentrations usedwere 0, 1 nM, and 10 nM and the nucleic acid/protein ratio was 1. Thebinding buffer used in this experiment was HBSMC, 0.01% HSA. Bindingreactions were incubated at 37° C. for 30 minutes, filtered through 0.45μm, nitrocellulose filters (15 mm), and then washed with 15 ml HBSMC.The RNA was recovered by phenol-urea extraction, eluted RNA was ethanolprecipitated in the presence of glycogen, resuspended in H₂O,supplemented with equal volume 2× formamide dye, and analyzed on 8%acrylamide, 8 M urea sequencing gels. Truncated RNAs that were bound toTGFβ2 were visualized and developed on a FUJIX BAS 1000 phosporimager(FUJI Medical Systems, USA).

[0083] Nucleic Acid Ligand Truncation

[0084] Truncated versions of full length nucleic acid ligands weregenerated in three ways. In one method, E. coli RNase H and hybrid2′-OCH₃ RNA/DNA oligonucleotides (5′N7 cleave, 3′N7 cleave; Table 1)were used to cleave nucleic acids at a specific site. Truncation SELEXis described in U.S. patent application Ser. No. 09/275,850, filed Mar.24, 1999, entitled “Truncation SELEX Method,” which is herebyincorporated by reference in its entirety. In a second method,overlapping DNA oligonucleotides encoding the desired ligand sequencewere annealed, extended by Klenow DNA polymerase, and then transcribed.In a third method, ligands were chemically synthesized with the desiredsequence.

[0085] Binding of Nucleic Acid Ligands to Human TGFβ's

[0086] The binding activity of individual ligands was determined bymeasuring the equilibrium dissociation constants using nitrocellulosepartitioning of labeled RNA as a function of protein concentration. RNAwas body-labeled or guanylated and then 5′-end labeled with γ³²P ATP andT4 polynucleotide kinase. Binding reactions were set at various proteinconcentrations (typically varied in either 3-fold or 10-fold increments)while maintaining the labeled RNA concentration constant at less than0.1 nM, and incubated at 37° C. for 10 minutes. Protein-RNA complexeswere partitioned away from uncomplexed RNA, by filtering the bindingreactions through a nitrocellulose/cellulose acetated mixed matrix (0.45μm pore size filter disks, type HA; Millipore, Co., Bedford, Mass.). Forfiltration, the filters were placed onto a vacuum manifold (12-well,Millipore, or 96-well BRL) and wetted by aspirating 1-5 ml of bindingbuffer. The binding reactions were aspirated through the filters, washedwith 1-5 ml of binding buffer and counted in a scintillation counter(Beckmann).

[0087] To obtain the monophasic equilibrium dissociation constants ofRNA ligands to hTGFβ2 the binding reaction:

[0088] KD

[0089] R:P→R+P

[0090] R=RNA

[0091] P=Protein

[0092] K_(D)=dissociation constant

[0093] is converted into an equation for the fraction of RNA bound atequilibrium:

q=(f/2R _(T))(P _(T) +R _(T) +K _(D)−((P _(T) +R _(T) +K _(D))²−4P _(T)R _(T))½)

[0094] q=fraction of RNA bound

[0095] P_(T)=total protein concentration

[0096] R_(T)=total RNA concentration

[0097] f=retention efficiency of RNA-protein complexes

[0098] The average retention efficiency for RNA-TGFβ2 complexes onnitrocellulose filters is 0.3-0.8. K_(d) values were obtained by leastsquare fitting of the data points using the software Kaleidagraph(Synergy Software, Reading, Pa.).

[0099] Competition between Ligands

[0100]³²P-labeled test ligands at a concentration of 1 nM were mixedwith increasing concentrations of unlabeled NX22283 (SEQ ID NO: 114).Then, an amount of TGFβ2 estimated to be near the K_(d) of the testligands was added (1 nM for NX22283, 1 nM for 21 a-21, 3 nM for 21a-4,and 10 nM for 14i-1). The reactions were incubated, filtered, washed andcounted as for a binding reaction.

[0101] Off-rate of NX22283

[0102] 1 nM ³²P-labeled NX22283 was mixed with 10 nM TGFβ2, incubatedfor 5 minutes to allow the protein to bind to the nucleic acid, and thena 1000-fold excess (1 μM) of unlabeled NX22283 was added. At varioustime points the reactions were filtered and washed to measure the amountof ³²P-labeled NX22283 that remained bound.

[0103] Biased SELEX

[0104] A library of sequences was constructed based on the sequence ofthe 34-mer truncate (NX22284) of nucleic acid ligand 21a-21. Thesequence of the DNA template (34N7.21a-21 (SEQ ID NO: 7)) is shown inTable 1. The randomized region is 34 bases long. At each position therandomized region consists of 62.5% of the NX22284 sequence and 12.5% ofthe other 3 nucleotides. Thus the randomized region is mutagenized ateach position (37.5%), but at the same time is biased toward thesequence of NX22284. The fixed regions (5′N7, 3′N7) were the same asused for the primary SELEXs.

[0105] To generate 34N7.21a-21 round 0 nucleic acid, the DNA templatewas amplified by PCR using the 5′N7 (SEQ ID NO: 2) and 3′N7 (SEQ ID NO:3) primers (Table 1). This PCR product was transcribed as describedabove in the filter partitioning SELEX section. This resulted in a34N7.21a-21 round 0 nucleic acid pool with the sequence shown in Table 1(SEQ ID NO: 10).

[0106] Filter partitioning as described above and in Fitzwater andPolisky (1996) (Meth. Enz. 267:275-301) with no competitors was used toenrich nucleic acids ligands that bound to human TGFβ2 the best. Theprotein concentration was reduced from ˜150-300 nM to 50 pM. The nucleicacid concentration was reduced from 1 μM to 1 nM. The nucleicacid/protein ratio ranged from 0.25 to 125. The round 5a pool of ligandswas cloned into pUC9 and sequenced as described above.

[0107] Bioactivity of TGFβ2 Nucleic Acid Ligands

[0108] The bioactivity of TGFβ2 nucleic acid ligands was measured withmink lung epithelial cells. Proliferation of these cells is inhibited byTGFβ2. Human TGFβ2 was titrated on the cells and ³H-thymidineincorporation was measured. The point at which ³H-thymidineincorporation by the cells was inhibited by 90-100% was determined(typically 1-4 pM). This inhibitory amount of TGFβ2 along with varyingamounts of nucleic acid ligand (typically 0.3 or 1 nM to 1 or 3 μM, in 3fold increments) was used. Typically, cells were plated at 10E5/ml in96-well plates in 100 μl MEM, 10 mM HEPES pH 7.4, 0.2% FBS. Following a4 hour incubation at 37° C., when cells were well attached to the wellsurface, TGFβ2 was added at 1-4 pM with or without nucleic acid ligandsas follows: the ligands were diluted across the 96 well plate in 3-folddilution steps and then TGFβ2 was added at 1-4 pM to all wells exceptcontrols. The cells were incubated for 16-18 hours prior to addition of³H-thymidine, and then incubation was continued for 20 additional hoursfollowing ³H-thymidine addition at 0.25 μCi per well. After incubation,the cells were lysed with 1% Triton X-100 and harvested onto GF/B filterplates in a Packard 96 well plate harvester, and ³H-thymidineincorporation in cellular DNA was quantitated by scintillation countingin MicroScint (Packard, Mariden, Conn.) in a Packard Top-Count. Datawere plotted as % of maximum ³H-thymidine incorporation vs RNAconcentration, and were fitted by the software Kaleidagraph (SynergySoftware, Reading, Pa.) to the equation m3*(m0+m1+(m2)−((m0+m1+(m2))*(m1+ml+(m2))−4*(mO)*((m2)))^ 0.5)/(2*(m2));where mO is the concentration of competitor RNA; ml is the IC50, m2 isthe concentration of TGFβ2, and m3 is the plateau value of the fractionof maximum ³H-thymidine incorporation. K_(i) values were determined fromIC₅₀ values according to the equation K_(i)=IC₅₀/(1+([T]/K_(dT)), where[T] is the molar concentration of TGFβ2 present in the assay and K_(dT)is the concentration of TGFβ2 causing 50% inhibition of MLECproliferation as determined by TGFβ2 titration experiments. This assaywas also used to determine the isotype specificity of RNA ligands wherethe three TGFβ isotypes were independently used as inhibitors of MLECreplication.

[0109] Pharmacokinetic Properties of NX22323

[0110] The pharmacokinetic properties of TGFβ2 ligand NX22323 weredetermined in Sprague-Dawley rats. NX22323 was suspended in sterile PBSand stored at ≦−20° C. Prior to animal dosing NX22323 was diluted withsterile PBS, to a final concentration of 0.925 mg/ml (18 μM, based onthe oligonucleotide molecular weight and the ultraviolet absorption at260 nm with an extinction coefficient of 0.037 mg of oligo/ml).Sprague-Dawley rats (n=2) were administered a single dose of NX22323 byintravenous bolus injection through the tail vein. Blood samples(approximately 400 μL) were obtained by venipuncture under isofluoraneanesthesia and placed in EDTA-containing tubes. The EDTA-treated bloodsamples were immediately processed by centrifugation to obtain plasmaand stored frozen <-20° C. Time points for blood sample collectionranged from 5 to 2880 minutes.

[0111] Standards and quality control samples prepared in blank ratplasma and plasma samples were analyzed by a double hybridization assay.To prepare plasma samples for hybridization analysis, 25 μL of plasmasample (or a dilution in plasma of the sample) was added to 100 μL of 4×SSC, 0.5% sarkosyl. A 25 μL aliquot was then mixed with 25 μL of 4× SSC,0.5% sarkosyl containing 24 μM capture oligonucleotide conjugated tomagnetic beads and 28 μM detect oligonucleotide conjugated to biotin ina covered 96-well microtiter plates. The mixture was allowed to incubateat 45° C. for 1 hour. Unbound oligonucleotide was removed and 0.1 ngstreptavidin alkaline phosphatase/μL NTT Buffer (0.8 M NaCl, 20 mM TrispH 7.5, 0.5% Tween 20) added to each well followed by a 30 minuteincubation at room temperature. The streptavidin alkaline phosphatasewas removed and the plate was washed twice with 200 μl NTT Buffer. TheNTT Buffer was removed and replaced with 50 μL DEA buffer (0.02% sodiumazide, 1 mM MgCl₂, 1% diethanolamine (Tropix, Inc., Bedford, Mass.), pH10). Then 34 μL/ml 25 mM chemiluminescent substrate for alkalinephosphatase (Tropix, Inc., Bedford, Mass.), and 20% Sapphirechemiluminescence amplifier (Tropix, Inc., Bedford, Mass.) in DEA buffer(50 μL/well) was added. The plate was incubated for 20 minutes at roomtemperature and read on a luminometer. A standard curve of NX22323 wasfit using a variable slope sigmoidal dose response non linear regressionequation (PRISM, version 2.00, GraphPad, San Diego, Calif.). Sample andquality control concentrations were extrapolated from the standard curveand corrected for dilution.

[0112] The average plasma concentration at each time point wascalculated and utilized in the pharmacokinetic analysis. Bothnoncompartmental and compartmental pharmacokinetic analysis were carriedout using WinNonlin version 1.5 (Scientific Consulting, Inc.). In thenoncompartmental analysis, the following parameters were calculated; themaximum concentration extrapolated at zero time (Cmax), the area underthe curve from zero to the last time point (AUClast), the area under thecurve from zero to infinite time (AUCINF), the terminal phase half life(Beta t1/2), the clearance rate (C1), the mean residence time calculatedfrom zero to infinite time (MRTINF), the volume of distribution atsteady state (Vss), and the volume of distribution during elimination(Vz). In the case of compartmental analysis, the following parameterswere calculated based on the minimum number of monoexponetial equationsto adequately fit the data: the maximum concentration extrapolated atzero time (Cmax), the area under the curve from zero to infinite time(AUCINF), the distribution phase half life (Alpha t1/2), terminal phasehalf life (Beta t1/2), the exponential constant for the distributionphase (A), the exponential constant for the terminal phase (B), theclearance rate (Cl), the mean residence time calculated from zero toinfinite time (MRTINF), and the volume of distribution at steady state(Vss).

Example 2 Isolation of Nucleic Acid Ligands That Bind Human TGFβ2

[0113] Several SELEX experiments on TGFβ2 have been attempted assummarized in FIG. 1. Several partitioning methods were applied atvarious stages of the SELEX progress including standard filtrationthrough nitrocellulose, spot, surface plasmon resonance biosensor(BIAcore), resonant mirror biosensor (Iasys), polystyrene beads, andpolyacrylamide gel shift. The combination of spot SELEX, surface plasmonresonance biosensor SELEX, and filter partitioning SELEX (withcompetitors) described here had the best overall improvement in affinity(˜>1000 fold) and thus is described in detail. In addition, a branch ofthis SELEX that utilized resonant mirror biosensor technology is alsodescribed.

[0114] Spot SELEX Conditions

[0115] Spot SELEX was chosen to initiate the SELEX process on humanTGFβ2 because it would allow a large amount of protein and nucleic acidto interact. The conditions used are shown in Table 3. The results ofround 1 were acceptable. The background was very low and the signal tonoise ratio was 5. At this point the population from the first round wasused for the surface plasmon SELEX in addition to continuing with thespot partition method. Ten rounds of spot SELEX were completed assummarized in Table 3 and a modest improvement in the affinity of thepool of about 30 fold was observed. These pools were not analyzedfurther.

[0116] Surface Plasmon Resonance Biosensor (spr) SELEX

[0117] Surface plasmon resonance biosensors were chosen as apartitioning medium because they provide very low background nucleicacid binding to the sensor, so that higher degrees of enrichment can beobtained. In addition binding and elution of nucleic acid can bemonitored and quantitated in real time.

[0118] TGFβ2 was coupled to a BIAcore biosensor using amine couplingchemistry. TGFβ2 coupled in this manner binds latency-associatedprotein, and recombinant soluble TGFβreceptors. FIG. 2 shows typicalsensograms obtained with LAP and recombinant sRIII where we observedk_(on) and k_(off) rates indicative of avid binding. During this SELEXexperiment as summarized in Table 4, a binding signal was first observedon the biosensor in round 6, increased up to round 9, and then decreasedin rounds 10 and 11. FIG. 3 shows sensograms with 0, 4-spr, 6-spr, 8-sprand 9-spr. FIG. 4 shows typical filter binding curves, in the presenceand absence of competitor tRNA, with representative pools from the sprSELEX and from these data it seems that round 9 binds in a biphasicmanner with a high affinity and low affinity K_(d) of 30 and 160 nM,respectively.

[0119] In bioactivity assays the K_(i) of the round 0 pool was about 2.6μM and the K_(i) of the round 9-spr pool was about 711 nM (see below).Sequence analysis of representative pools indicated that such poolsmaintained significant complexity up to round 8 while after round 9 suchpools were strongly biased towards a single sequence.

[0120] Round 8-spr was cloned and sequenced. A total of 69 clonesrepresenting 51 different sequences were analyzed. Four sequences (#8.2, 8.3, 8.9 and 8.48; see Table 5) were represented more than once andaccounted for 21 of the 69 clones. All four of these sequences boundTGFβ2 and were 2 or 3 base variants of a clone (14i-1) isolated from thefilter SELEX (see below). Twenty three other sequences were nonbindingor filter-binding sequences (see Table 5) and 25 clones were not testedfor binding.

[0121] Resonant Mirror (rm) Optical Biosensor SELEX

[0122] Since the affinity of nucleic acid pools selected on the surfaceplasmon resonance biosensor peaked at round 9-spr, resonant mirror (rm)optical biosensor technology was tested to determine if it could advancethe affinity of nucleic acid ligands any further. Resonant mirroroptical biosensor technology offers many of the same advantage assurface plasmon biosensor technology, but in addition the binding isdone in a cuvette under equilibrium conditions rather than over thesurface of a chip under flow conditions. Within the cuvette the bindingcan be extended for long time periods. Therefore the nucleicacid/protein binding reaction can be more stringent and selective.

[0123] For resonant mirror SELEX, TGFβ2 was coupled to two biosensorcuvettes using amine coupling chemistry. In one cuvette the TGFβ2 wasinactivated by SDS denaturation and this cuvette served to assessbackground. The other cuvette containing active TGFβ2 was used for theSELEX. Beginning with round 9-spr, five rounds were done using resonantmirror optical biosensor technology. The conditions used for and theresults of the resonant mirror SELEX are shown in Table 6.

[0124] Biosensor signals were observed for each round. The binding ofthe nucleic acid pools from rounds 10-rm to 12-rm was assessed (Table 6;FIG. 5). The pool K_(d) improved modestly up to round 12-rm with nofurther improvement in the subsequent rounds. The round 12-rm pool bindsbiphasically with high and low affinity K_(d) of ˜2nM and ˜150 nM,respectively.

[0125] In bioactivity assays the K_(i) of the round 13-rm pool was about505 nM. Round 13-rm was chosen for subcloning and sequence analysis. Of15 clones that were sequenced, all 15 (Table 7) were 1 to 5 basevariants of a clone (14i-1) which was originally isolated from thefilter SELEX (see below).

[0126] Filter Partitioning SELEX

[0127] Round 8-spr was used as the starting material for a filter SELEX.The properties of round 8-spr were studied and it was found that 1) asignificant fraction bound to a nitrocellulose filter (10%), 2)significant nucleic acid binding (defined here as signal/noise>2) toTGFβ2 was not detectable using nucleic acid-excess conditions, and 3) inthe presence or absence of polyanionic competitors there was asignificant decrease in the binding of round 0, but not round 8-spr toTGFβ2. These findings had implications that are addressed below.

[0128] Use of a Competitor

[0129] The binding of round 8-spr nucleic acid to TGFβ2 in the presenceof a polyanionic competitor (yeast tRNA) was studied at various ratiosof competitor to nucleic acid. It was found that a 75,000 fold excess oftRNA over round 8-spr nucleic acid resulted in 50% inhibition ofbinding, whereas a 6,000 fold excess of tRNA over the round 0 nucleicacid pool resulted in 50% inhibition of binding. Heparin also competedwith RNA for binding to TGFβ2, but about 10-fold more heparin was neededto inhibit RNA binding to TGFβ2 to the same degree as that observedusing tRNA. By including a 100,000-fold excess of yeast tRNA over RNA ina TGFβ2/RNA binding experiment, a 100-fold difference in binding betweenround 0 and round 8-spr was detected, whereas a 3-fold difference wasobserved in the absence of any competitor. Thus in the presence of anappropriate amount of competitor, the binding of selected nucleic acidpools is unaffected, whereas the binding of round 0 nucleic acid isreduced substantially. When competitors are not included in studying thebinding of TGFβ2 to nucleic acid the affinity of nucleic acids selectedusing the SELEX process can be grossly underestimated. In this regardTGFβ2 is similar to other “professional” nucleic acid binding proteins(e.g., restriction enzymes, polymerases, transcription factors, etc.),in that it possesses both a low affinity, nonspecific and a highaffinity, specific nucleic acid binding activity. The difference betweenthese 2 binding modes can be revealed in the presence of competitors.Competitors are often used in the study of transcription factors. Forexample, it can be difficult to detect specific binding of a crudeextract containing a transcription factor to oligonucleotidesrepresenting their cognate site in gel-shift experiments, unless acompetitor, such as poly [dI-dC]  poly [dI-dC], is included in thebinding reaction.

[0130] Nonspecific binding can involve the binding of multiple proteinsper nucleic acid, often at low affinity sites, giving a false appearanceof high affinity. A protein can bind at multiple sites on a nucleic acidor protein aggregates may form on a single protein bound to a nucleicacid. TGFβ2 is well known to be “sticky.” In the absence of a competitorof nonspecific interactions, TGFβ2 may form large networks and complexesof nucleic acid and protein involving primarily nonspecificinteractions. Gel shift analysis of TGFβ2, in the absence of competitor,supports these ideas because TGFβ2 does not form distinct (one to one)complexes with nucleic acid in gels, but instead either remains in thewell at the top of a gel or forms smears that may represent largeheterogeneous nucleic acid/protein complexes.

[0131] Besides the implications for doing binding curves, the nucleicacid-binding properties of TGFβ2 may have implications for SELEX. Forexample the high level of nonspecific binding of nucleic acid by TGFβ2may have interfered with previous SELEXs by obscuring specificinteractions or preventing the isolation of nucleic acid/proteincomplexes that involved only specific binding interactions. That is, ifmixtures of specific and nonspecific nucleic acid interactions exist innucleic acid/TGFβ2 complexes that form, then the selection for specificinteractions may be difficult, if the nonspecific interactions are noteliminated. Lack of progress in some previous SELEX experiments may havebeen due to efficient competition by the large excess (>10¹²-10¹⁴) oflow affinity nucleic acids that contain nonspecific binding sites with asmaller number (˜10-1000) of high affinity nucleic acids that containspecific binding sites, especially in early rounds of SELEX.

[0132] Use of Protein-excess or Nucleic Acid-excess Conditions

[0133] Given the discussion above, a question arises as to which type ofSELEX condition is better for a protein such as TGFβ2, protein-excess ornucleic acid-excess. Protein-excess conditions may tend to encouragenonspecific interactions. However as long as the competitor/nucleic acidratio is high enough to eliminate enough nonspecific interactions, butretain specific interactions, this may not be an issue. One advantage tousing protein excess is the bound to background ratios are better andbackground is lower, which would results in better levels of enrichment.

[0134] Nucleic acid-excess conditions may not discourage nonspecificinteractions because within nucleic acid pools used for SELEX the ratioof nonspecific to specific binding nucleic acids (which is what is mostimportant) would be the same no matter what the nucleic acidconcentration is. In addition nucleic acid-excess would reduce thecompetitor/nucleic acid ratio which would tend to increase nonspecificinteractions. As discussed above the ratio of tRNA to nucleic acid mustbe at least 100,000 in early round of the filter SELEX in order foraffinity enrichment to be efficient. This can be technically difficultin early rounds of SELEX when the nucleic acid concentration istypically higher. One advantage to using excess nucleic acid is thatmore members of a given sequence would be represented in a pool. Howeverif there had been enough enrichment (e.g., using a method such assurface plasmon resonance SELEX) prior to filter SELEX there willprobably be multiple representatives of a given sequence and this wouldnot be an issue.

[0135] Filter SELEX Conditions

[0136] The conditions used at each round of the filter SELEX are shownin Table 15. Multiple conditions (up to 12) were used in each roundvarying nucleic acid/protein ratios, competitor/nucleic acid ratios,filter washing buffers, and filter washing volumes. Typically conditionsthat resulted in the lowest background (<1%) and a significantbound/background ratio (>2) were processed for the next round. Only datafor SELEX rounds that were used in the next round are shown in Table 15.

[0137] The SELEX began by using an amount of tRNA competitor(100,000-fold excess) that was determined in the SELEX reaction toinhibit binding of round 8-spr to TGFβ2 by about 60%. SELEX reactionswith competitor were done for round 9b through 14i. The inclusion oftRNA in round 9 also dramatically reduced binding of round 8-spr nucleicacid to nitrocellulose filters from ˜10-15% to ˜1%. The higher the“background” binding is in a SELEX reaction, the lower the maximumpossible enrichment. Thus inclusion of tRNA in the early rounds of thefilter SELEX may have had a dual benefit. It not only may haveeliminated nonspecific binding of TGFP2 to nucleic acid, but alsoallowed more enrichment by reducing background. At round 15c loweraffinity competitors were no longer effective at reducing binding ofnucleic acid and were not used. This is presumably because the nucleicacid pool bound TGFβ2 with adequate specificity and affinity. Thereforefrom round 16 to 22, the presumed specific nucleic acids were allowed tocompete with each other by using more traditional nucleic acid-excessconditions.

[0138] The background increased to unacceptable levels in rounds 15c and16a. Gel shift partitioning was investigated as an alternativepartitioning procedure at this point but did not work. By modifying thewashing conditions the background was reduced to 0.2% in round 17a.After round 18b it was possible to do SELEX rounds at proteinconcentrations below 1 nM and under nucleic acid-excess conditions. Itwas also found that nucleic acid concentrations above 1-5 nM also helpedto reduce background in some rounds.

[0139] In summary, during the filter SELEX, the concentration of theprotein was reduced 30,000-fold, from 300 nM in round 9b to 10 pM inround 22a. The background binding to filters was reduced from 10% to0.1%. Nucleic acid pools that bound to TGFβ2 only when protein-excessconditions (˜100 protein/1 nucleic acid) were used were selected to bindunder high nucleic acid/protein (>100/1) or competitor/nucleic acid(>10⁷/1)-excess conditions.

[0140] Binding of Nucleic Acid Pools from Filter SELEX

[0141] The binding of TGFβ2 to selected nucleic acid pools improvedsteadily, but slowly and erratically. There was an improvement in thebinding of round 10b (K_(d)=˜100 nM) compared to the starting pool(round 8-spr; K_(d)˜500 nM). The affinity of round 1 a was the same as10 b and that of round 12d improved modestly to ˜40 nM. Rounds 13i and15c bound TGFβ2 approximately the same (K_(d)=˜30), while round 14i mayhave bound worse (K_(d)=˜75). Round 16a nucleic acid (K_(d)=˜10 nM)bound slightly better than round 15c. There was ˜2-fold improvement inaffinity of the nucleic acid pool from rounds 16a to 17a (K_(d)=˜5 nM).The K_(d) of round 18a nucleic acid (˜5 nM) was equivalent to round 17anucleic acid. There was another slight increase in affinity from round18b to 19a (K_(d)=˜2-3 nM). The affinity of rounds 20a, 21a and 22aplateaued at about 1 nM. The SELEX was stopped at round 22a because thebound to background ratio was below 2 and it would have been technicallydifficult to reduce the protein concentration to 1-3 pM in round 23.

[0142] In summary the K_(d) improved from ˜500 nM in round 9b to ˜1 nMin round 21 a, resulting in an overall improvement of ˜500-fold in thefilter SELEX and >10,000-fold in the entire SELEX. The averageimprovement per round was only about 1.6-fold. This rate of improvementis slow compared to an average SELEX experiment, which may take ˜5rounds using only surface plasmon resonance technology or ˜10 roundsusing only filter partitioning.

[0143] Inhibition of Bioactivity by Nucleic Acid Pools

[0144] Rounds 0, 9-spr, 13-rm, 14i, 18b and 19a, and latency-associatedprotein (LAP) were tested on mink lung epithelial cells for theirability to reverse TGFβ2-mediated inhibition of ³H-thymidineincorporation. The results are that the K_(i) of the round 9-spr poolwas about 711 nM. The K_(i),s of the round 14i, 18b, 19a and 21a poolswere about 231 nM, 309 nM, 154 nM and 10 nM, respectively. The K_(i), ofLAP was about 0.5 nM.

[0145] From these results it can be concluded that inhibitors of TGFβ2were enriched in the later rounds of the TGFβ2 SELEX. In addition thereis a continuous correlation between the affinity measured in vitro andthe inhibitory activity measured in vivo:

[0146] LAP <round 19A<round 14i <round 13-rm <round 8-spr <round 0.

[0147] Sequencing of Nucleic Acid Ligand Clones Isolated from FilterSELEX

[0148] Based on several criteria (pool K_(d), filter-binding background,bound to noise background, inhibitory activity in cell assay, andabsence of aberrant products during the RT-PCR steps of SELEX) round 21awas subcloned for sequence analysis. Forty eight clones were sequencedfrom round 21a (Table 8). Two unique sequences represented by clones21a-4 and 21 a-21 (the first number refers to the SELEX round a clonewas initially isolated from and the second number is a clone number)were identified. Several clones were minor variants (1-6 basesdifferent) of clones 21a-4 and 21a-21. One hundred more clones werescreened by PCR using primers specific for clones 21 a-4 and 21 a-21. Ofthese, 90 were clone 21 a-21-like, 9 were clone 21a-4-like and 1 was athird unique sequence (21a-48), which was shown to be a nitrocellulosefilter-binding sequence. In conclusion, round 21 a consists almostentirely of two sequences and variations of those sequences. This wasnot surprising because round 21 a was the second to last round and thebulk affinity of the nucleic acid pools had not improved much from round19a to 21 a.

[0149] Since the sequence diversity of round 21 a was restricted, 3other rounds (14i, 16a, 18a) were also sequenced. Only one more novelsequence (14i-1 and variants) was isolated. Two filter-binding sequenceswere also isolated (16a-1 and a variant of 21a-48). Therefore, as withrounds 8-spr, 13-rm and 21a, these 3 rounds also did not contain diverseTGFβ2-binding nucleic acid ligands.

[0150] The sequences of 14i-1, 21 a-4, and 21 a-21 are shown in Table 8.The affinity of the sequences for human TGFβ2 is about 10 nM, 3 nM and 1nM respectively. Therefore these 3 sequences are ligands that bind humanTGFβ2 with high affinity.

[0151] The ligands were tested for inhibitory bioactivity. The K_(i)'sof 14i-1, 21 a-4 and 21 a-21 are about 200 nM, 30 nM and 10 nMrespectively. Thus these ligands are also inhibitory ligands. As for thepools the binding affinity correlates well with the inhibitory activity.This is not surprising since it is likely the TGFβ2 ligands bind nearthe heparin binding site which is very close to the TGFβ receptorbinding region. The inhibitory activity of ligand 21a-21 was alsocompared to that of antibodies.

[0152] Clones were isolated and sequenced from six rounds (8-spr, 13-rm,14i, 16a, 18a and 21a). The number of each type of sequence issummarized Table 9. Out of 264 clones analyzed by sequencing and 100clones analyzed by a PCR-based analysis using ligand-specific primers(Table 10), only 3 different TGFβ2 ligand sequences (and minor variants)were obtained. Fifteen sequences were filter binding sequences and 36were nucleic acids that do not bind well to filters or TGFβ2. The degreeof restriction in sequence diversity observed in this SELEX is veryunusual. Generally one can isolate dozens of different nucleic acidligands and usually it is possible to find high affinity rounds were oneligand represents <10% of the population.

[0153] Since sequencing and screening of 6 rounds of SELEX that are asmuch as 13 rounds apart did not result in a diverse set of sequences theproperties of the pools were investigated further to determine where thesequence diversity was restricted. Selected nucleic acid pools weresequenced and semi-quantitative RT-PCR on nucleic acid pools usingligand-specific primers was done. The results are shown in (Table 10).Taken together with the sequencing results, it appears that arestriction in sequence diversity during the SELEX process may haveoccurred near rounds 6-spr or 7-spr.

[0154] Clone 14i-1 is first detectable in round 6-spr, becomes mostfrequent near round 14i, and decreases in frequency in later rounds.Clone 21 a-4 is first detectable by sequencing in round 14i, is mostabundant in round 16a, and decreases in frequency by round 21a. However21a-4 may exist in prior rounds (RT-PCR analysis of pools using a primerspecific for clone 21 a-4 was not done.). Clone 21a-21 was rare in round14i (<1/104 clones by sequencing; estimate <1/200-500 clones by RT-PCR),became more frequent in round 16a, and composes most of round 18b and21a.

[0155] It appears the surface plasmon resonance biosensor SELEX resultedin a high degree of diversity restriction, which has been observedbefore using this technology. The reason why various later rounds wouldconsist of virtually one sequence is not clear. Perhaps only a verysmall number of sequences bind TGFβ2 under the selection conditionsused. Perhaps a change in selection conditions such as the inclusion ofcompetitors at round 9 or the switch from protein-excess bindingreactions to nucleic acid-excess binding reactions at round 16 resultedin the emergence of clone 21 a-21 as the predominant clone by round 21a. It seems as though the selection pressures were significant becausethe predominant ligand in a pool changed in as few as 2 rounds.

[0156] The pattern of changes in the population of nucleic acid ligandscan be explained by analogy to the theory of natural selection. In anearly SELEX round, a variety of sequences will exist. Strong selectivepressure may narrow the sequence variation considerably, to the pointthat a single sequence is predominant. However rare ligands still existthat can be selected in future rounds or during significant changes inselective pressure. This is true in any SELEX experiment, but the TGFβ2SELEX experiment described here may be an extreme example. In spite ofthe restriction on sequence diversity, better binding ligands couldeventually be isolated. Note that ligand 21 a-21 was first identified bysequencing in round 16a. Thus rare, high affinity nucleic acid ligandsmay exist even in round 22 that would only become predominant under thecorrect selection conditions. One approach for isolating such raresequences might be to specifically deplete late rounds of SELEX of knownsequences (e.g., by hybrid selection, restriction enzyme digestion ofPCR products, site-directed RNase H cleavage of nucleic acid), anapproach that this TGFβ2 SELEX is well suited for since essentially only5 different sequences (3 ligands and 2 filter binding sequences) werepresent in later rounds. Isolating a sequence that is present in <1/1000clones might be easy using depletion methods, but would be tedious usingsequencing or PCR screening methods.

[0157] These results raise questions about when a SELEX is done and howto judge whether it is done. In this SELEX, standard criteria forjudging when a SELEX is done such as K_(d) improvement, and sequencingof clones or bulk nucleic acid pools may not be good criteria forjudging if the SELEX had proceeded as far as it could. Often there aretechnical limitations (background, reaction volumes, loss of low amountsof protein to large surfaces) that determine when a SELEX must beterminated and these are artificially limiting. Perhaps a “depletionSELEX” round should be done at the end of every SELEX to attemptenrichment of ligands that would be difficult to isolate by currentlyused methods.

[0158] Specificity of Human TGFβ2 Ligands

[0159] For nucleic acid ligands to be most useful in the applicationsclaimed herein they should be highly specific for a particular subtypeof TGFβ. The specificity of human TGFβ2 ligands was investigated by inseveral ways.

[0160] The specificity of TGFβ2 ligands was examined using the cellculture bioactivity assay where the specificity of the TGFβ2 (describedhere) and TGFβ1 (see U.S. patent application Ser. No. 09/046,247, filedMar. 23, 1998, entitled “High Affinity TGFβ Nucleic Acid Ligands andInhibitors,” now U.S. Pat. No. 6,124,449, which is incorporated hereinby reference in its entirety) aptamers was compared to the specificityof antibodies. Two types of antibodies were used namely, monoclonalantibodies and immunopurified polyclonal antibodies. It was found (FIG.6) that the TGFβ2 ligand NX22283 inhibited TGFβ2 protein bioactivity(K_(i=)=10 nM), but not TGFβ1 (K_(i=)>1000 nM) or TGFβ3 bioactivity(K_(i=)>1000 nM). The TGFβ2 ligand NX22283 inhibits the TGFβ2bioactivity with a potency equivalent to that of a monoclonal antibodywhile the most potent inhibitor of TGFβ2 bioactivity in this experimentwas an affinity-purified polyclonal antibody.

[0161] The specificity of a TGFβ2 ligand for TGFβ2 compared to TGFβ3 wasalso analyzed in nucleic acid binding assays. The affinity of round 040N7 nucleic acid or the full-length TGFβ2 ligand 21a-21 to human TGFβ2protein was >10 μM or 1 nM, respectively. The affinity of round 0nucleic acid or ligand 21a-21 to human TGFβ3 protein was >10 μM or >30μM, respectively. Therefore the TGFβ2 ligand does not bind significantlyto TGFβ3.

[0162] It was found that the TGFβ1 ligand 40-03 (1 (see U.S. patentapplication Ser. No. 09/046,247, filed Mar. 23, 1998, entitled “HighAffinity TGFβ Nucleic Acid Ligands and Inhibitors,” now U.S. Pat. No.6,124,449), bound to TGFβ3 although 1000-fold worse. These resultsindicate there may be one or more amino acids in common between TGFβ1and TGFβ3 that are not found in TGFβ2 so that a TGFβ1 ligand can bindTGFβ1 and TGFβ3 but not TGFβ2 and so that the TGFβ2 ligand 21a-21 bindsTGFβ2 but not TGFβ1 or TGFβ3. Indeed, as shown in Table 12, there are 19amino acids out of 122 that are found in TGFβ2, but not in TGFβ1 orTGFβ3. Three of these differences (Lys-25, Arg-26, and Lys-94 in TGFβ2)are within a putative heparin binding region and may be important fordetermining the binding specificity of TGFβ ligands.

[0163] Truncation of Nucleic Acid Ligands

[0164] It is desirable to obtain the smallest “truncate” of a fulllength nucleic acid ligand so that it can be synthesized efficiently atthe least cost. The goal of this study was to obtain ligands that areless than half their original length (<35 bases), yet retain about thesame affinity as the full length ligand. Several approaches were used toidentify truncates of the three TGFβ2 ligands.

[0165] RNase H and hybrid 2′-OCH₃ RNA/DNA oligonucleotides (5′N7 cleave,3′N7 cleave; Table 1) were used to remove the 5′ and 3′ fixed sequencesfrom 2′-F pyrimidine, 2′-OH purine nucleic acid ligands as described inU.S. patent application Ser. No. 09/275,850, filed Mar. 24, 1999,entitled “Truncation SELEX Method,” which is hereby incorporated byreference in its entirety.

[0166] Second, the “boundaries” of the ligands were identified using apreviously described method (Fitzwater and Polisky (1996) Met. inEnzymol. 267:275-301). Boundaries define the 5′ and 3′ ends of thesmallest truncate. However boundary determination does not identifyinternal deletions that can be made. Also because of the nature of theboundary determination method, if a boundary falls within a run ofpyrimidines or is too close to either end, then which nucleotide is theboundary must be determined by other methods (e.g., generation ofligands beginning or ending with each candidate boundary positionfollowed by analysis of their binding to TGFβ2).

[0167] A third method used relied on plausible structural motifs todefine hypothetical sequence boundaries. Synthetic oligonucleotidescorresponding to these boundaries were synthesized and were tested forbinding to TGFβ2.

[0168] A fourth approach for identifying TGFβ2 ligand truncates was tolook at the location of sequence variations in each ligand. In ligands21 a-4 and 21 a-21 the changes that occurred in sequence variants weredistributed randomly throughout their sequences. However in ligand14i-1, the sequence changes in variants were highly localized. Thisimplied that the variable region of ligand 14i-1 could tolerate changeswithout affecting binding and that the whole variable region may bedispensable. A fifth approach was to make internal deletions based onpredicted structures. Portions of putative bulges, loops, or basepair(s) within predicted stems can be deleted. The success of thismethod depends critically on how close the structural model is to thereal structure. For 21 a-21 the most stable structure was found to beincorrect. Only when a structure closer to the real structure wasidentified (by using the biased SELEX method) could internal deletionsof 21 a-21 successfully be made.

[0169] Truncation of Ligand 14i-1

[0170] Using the RNase H truncation method it was determined that ligand14i-1 requires the 5′ but not the 3′ fixed sequence (Table 13).Consistent with this result, when both the 5′ and 3′ fixed sequenceswere removed, ligand 14i-1 did not bind TGFβ2.

[0171] Conventional boundary experiments defined the 3′ end of ligand14i-1 (FIG. 7) to be within positions 39-45. In the same experiment wefailed to observe a clear boundary at the 5′ end of this ligand. Of the60 sequence variants of ligand 14i-1, 54 have nucleotide changes thatoccur within the last 16 bases at the 3′ end of the selected sequenceregion. Most of the variants have single base changes, but a few have asmany as 6 bases changed. Such changes may or may not affect binding. Ifthey affect binding then that region is important for binding. Morelikely, since there are so many changes in so many clones within thatregion, those bases are probably not important. It was surmised they maybe able to be deleted, possibly along with the adjacent 3′ fixed region,without affecting binding. This idea was confirmed by twoexperiments: 1) The binding of 8 sequences that varied within the 16base region and had different sequence changes was tested. They allbound as well to TGFβ2 as the 14i-1 ligand. 2) A 38 base long truncateof 14i-1 (14i-1t5-41; Table 13) that lacked the 3′ fixed region and the16 base variable region bound to TGFβ2 as well as the full length (71base long) ligand.

[0172] Four sequences that removed additional bases from the ends of14i-1 beyond those removed in 14i-1t5-41 were made [(14i-1t5-38,14i-1t5-35, 14i-1(ML-87), and 14i-1(ML-89]. Also one internal deletionof 14i-1t5-41 was made [14i-1(ML-86)]. None of these bound to TGFβ2(Table 13). Taken as a whole these experiments showed that theboundaries of ligand 14i-1 fall within positions 5-7 at the 5′ end and39-41 at the 3′ end. All these results defined a truncate for ligand14i-1 that is 38 bases long (Table 13).

[0173] Truncation of Ligand 21a-4

[0174] Using the RNase H truncation method it was determined that ligand21 a-4 requires the 5′ but not the 3′ fixed sequence (Table 14). Whenboth the 5′ and 3′ fixed sequences were removed ligand 21a-4 did notbind TGFβ2.

[0175] The boundaries of ligand 21a-4 (FIG. 7) are at positions U 11 inthe 5′ fixed region and within positions 52-56 on the 3′ end, defining atruncate that is between 42-46 bases long (Table 8). This is consistentwith RNase H truncation results which show 21 a-4 requires the 5′ endbut not the 3′ end to bind TGFβ2.

[0176] By examining hypothetical structures, the boundaries for 21 a-4were predicted to occur at position G12 at the 5′ end and position C48at the 3′ end. These positions agree well with the region defined by theboundary method. A 37 base long truncate of 21a-4 (excluding sequencesrequired to initiate transcription), beginning at position 12 and endingat position 48 [(21 a-4(ML-110); Table 14)], bound as well to TGFβ2 asthe full length 21a-4 ligand.

[0177] One sequence that removes 4 additional bases from the 3′ end of21 a-4(ML 110) was made that is called 21a-4(ML-111). The binding of21a-4(ML-111) was reduced 30-fold compared to 21a-4(ML-110). Also threeinternal deletions of 21a-4(ML-110) were made [21a-4(ML-92, ML-108, andML-109)]. None of these bound well to TGFβ2 (Table 14). A sequence[21a-4(ML-91)] that added 2 bases to the 3′ end of 21a-4(ML111) did nothave any improved binding compared to 21 a-4(ML-110). Thus, the smallesttruncate of ligand 21 a-4 identified, that retains binding, is 42 baseslong.

[0178] Truncation of Ligand 21a-21

[0179] Using the RNase H truncation method it was concluded that ligand21a-21 requires the 3′ but not the 5′ fixed sequence (Table 11).However, when both the 5′ and 3′ fixed sequences were removed, ligand21a-21 bound TGFβ2. This seems paradoxical since removal of the 3′ endalone eliminates binding. However, the data can be interpreted to meanthat the 3′ deletion folds in a structure that does not bind to TGFβ2,while the truncate that lacks both ends does not fold into a dead endstructure. Indeed Mfold structure predictions indicate this may be thecase.

[0180] The boundaries of ligand 21a-21 (FIG. 7) are at position G21 onthe 5′ end and within positions 50-55 on the 3′ end, defining a truncatethat is 30-35 bases long. The results are consistent with RNAse Htruncation data which shows that 21 a-21 requires neither the 5′ nor the3′ end to bind TGFβ2. The truncate identified by boundaries fallscompletely within that defined by RNase H truncation.

[0181] Synthetic sequences based on putative structures were also testedas summarized in Table 11. Results from these experiments are inagreement with the RNAse H and conventional boundary experiments.

[0182] Several additional end truncates and internal deletions of 2 a-21(ML-95) were made (Table 11). The 9 end truncates included21a-21(ML-96), 21a-21(ML-97), 21a-21(ML-103) 21a-21(ML-104)21a-21(ML-105), NX22286, NX22301, NX22302, and NX22303. Of these onlyNX22301, which removes one base at the 5′ end, binds as well as21a-21(ML-95). Internal deletions included 21a-21 (ML-99), 21a-21(ML-101), 21a-21(ML-102), 21a-21 (ML-114),21a-21(ML-115),21a-21(ML-116),21a-21(ML-118),21a-21(ML-120),2a-21(ML-122),21a-21(ML-128),21a-21(ML-132), 21a-21(ML-134), 21a-21(ML-136), and 21a-21(ML-138). Ofthese 14 internal deletions, only 21a-21(ML-130) bound about as well as21a-21(ML-95).

[0183] Three sequences [21a-21(ML-94), NX22283, and NX22285), were madethat are longer than 21a-21(ML-95). Of these only NX22285 may have bound(marginally) better than 21a-21(ML-95). Thus, the shortest ligandidentified that binds TGFβ2 is the 34-mer NX22284.

[0184] NX22283 and NX22284, which are synthetic analogs of thetranscribed ligands 21 a-21(ML-94) and 21a-21(ML-95), respectively,bound with identical affinity to TGFβ2 (Table 11). The synthetic nucleicacids also have the same inhibitory bioactivity as their transcribedanalogs; on the other hand, the short, 30-base long NX22286 and itstranscribed analog 21a-21 (ML-96) do not bind TGFβ2 and they do notinhibit TGFβ2 bioactivity. Therefore synthetic nucleic acids have thesame properties as their transcribed counterparts.

[0185] To summarize, truncated 14i-1, 21a-4, and 21a-21 ligands wereidentified that bind TGFβ2 as well as the full length ligands and are38, 37 (excluding 5 bases added to improve transcription yield), and 32bases long, respectively. Twenty four sequences were made in an attemptto shorten the NX22284 truncated ligand (8 single base deletions and 16multiple base deletions). Only 2 of them, (21a-21(ML-130) and NX22301,bind to TGFβ2. Therefore it appears that the sequence and spacing ofstructural elements in NX22284 must be maintained for binding to occur.

[0186] Competition between Ligands for Binding TGFβ2

[0187] Examination of the ability of different ligands to compete witheach other for binding to a protein can indicate whether the ligandsbind to a similar (or overlapping) or distinctly different regions onthe protein.

[0188] The ability of NX22283, a truncate of ligand 21 a-21 (Table 14),to compete with 4 ligands (14i-1, 21a-4, 21a-21, and NX22283) wastested. The results were that NX22283 competed best with itself, thenwith 21a-21, 21a-4, and 14i-1, in decreasing effectiveness. Thus, theability of NX22283 to compete correlates with how related its sequenceis to the sequence of the test ligand. NX22283 is most closely relatedto itself and competes best with itself. NX22283 is a truncate of21a-21, and competes with 21a-21 second best. The sequence of 21a-4 maybe distantly related to 21a-21 and NX22283 competes with 21a-4 thirdbest. The sequence of ligand 14i-1 is not related to NX22283, andNX22283 is least capable of competing with 14i-1.

[0189] The concentration range of NX22283 required to inhibit 50% of thebinding of the other ligands was 10-fold. Since these differences in theamount of NX22283 it took to compete off the other ligands can beattributed to differences in their affinity there is probably only onetype of binding region for these ligands on TGFβ2. However, there may beone or more similar sites per homodimer of TGFβ2. If there were twodistinct types of nucleic acid binding sites on TGFβ2 (as is the casefor the HIV-1 gag protein; Lochrie et al. (1997) Nucleic. Acids Res.25:2902-2910) it should take >1000 times as much competitor (i.e., thedifference between the K_(d) of round 0 nucleic acid and the K_(d) ofNX22283) to compete off a ligand binding at a second distinct site,because presumably a ligand that has high affinity at one site wouldhave low affinity for a distinct site. This was not observed.

[0190] Off-rate of NX22283

[0191] The half-life for NX22283-TGFβ2 complex was measured in 2experiments to be 0.5 or 3 minutes. Almost all of the ligand dissociatedfrom TGFβ2 in 60-75 minutes. Although these times may seem short, theyare typical of in vitro off-rate measurements for nucleic acid ligandsthat have been isolated by filter partitioning SELEX.

Example 3 Nucleic Acid Ligands Isolated by the SELEX Method using aBiased Round 0 Library

[0192] A biased SELEX is one in which the sequences in a nucleic acidpool are altered to bias the result toward a certain outcome. Theprimary goals of a “biased” SELEX are to obtain ligands that have ahigher affinity and to determine what the putative secondary structureof a ligand may be. The starting, round 0 nucleic acid library (called34N7.21a-21) used for the TGFβ2 biased SELEX had the same 5′ and 3′fixed regions (5′N7 and 3′N7) as the prior TGFβ2 SELEX (Table 1). It wasmade as a 2′-F pyrimidine, 2′-OH purine nucleic acid. However, asdescribed in the “Materials and Methods” section, the random region was34 bases long. Within the randomized region 62.5% of the nucleotides ateach position correspond to the NX22284 sequence. The remaining 37.5%correspond to the other three nucleotides. Thus each position ismutagenized and the sequence of the pool is biased toward the NX22284sequence. Selection for ligands that bind to TGFβ2 using such a poolshould allow variants of NX22284 to be isolated, some of which may nothave been present in the original 30N7 round 0 pool.

[0193] The bulk K_(d) of the round 0 34N7.21a-21 pool was about 870 nM(Table 15) using protein-excess binding conditions. This is at least10-fold better than for the unbiased round 0 40N7 pool, as would beexpected. This round 0 nucleic acid pool also bound under nucleicacid-excess conditions in small scale SELEX type reactions, althoughpoorer than in protein-excess reactions, as would be expected. Theprogress of the biased SELEX is shown in Table 15.

[0194] The conditions used in the biased SELEX and the results are shownin Table 15. A total of 9 rounds were done. Attempts were made to obtainhigher affinity ligands by using competitors, starting at round 4. Bothyeast tRNA (low affinity) and NX22284 (high affinity) were used ascompetitors. Both are nonamplifiable during the PCR step of SELEX. The“A” series was done without competitors while the “B” series was donewith competitors.

[0195] The binding of the nucleic acid pools to TGFβ2 was measured forrounds 0 to 8 (Table 15) and found to improve from ˜870 nM for the round0 nucleic acid library to ˜1 nM for the round 5a nucleic acid pool.Competition seemed to have little consistent effect on affinityimprovement in this SELEX experiment. Probably competition should havebeen initiated with NX22284 at round 1. Peak improvement in the poolaffinity plateaued in rounds 5, 6 and 7, and 8. Therefore round 5a, theearliest round with the best affinity, was subcloned and sequenced.

[0196] Sequences of TGFβ2 Nucleic Acid Ligands obtained from a BiasedSELEX.

[0197] As shown in Table 16, 25 unique sequences were obtained. One tonine changes from the starting sequence were found. All of the cloneswere 34 bases long within the selected sequence, consistent with studies(see “truncation of ligand 21 a-21 ” above) where it was difficult todelete any internal bases.

[0198] Covariance between pairs of positions was analyzed by eye and byusing the consensus structure matrix program (Davis et al. (1995)Nucleic Acids Research 23:4471-4479). Covariance was observed between 2different areas implying the existence of 2 stems in the structure. Thepattern of covariance suggests the structural model shown in FIG. 8 or asimilar variant of that structure (e.g., some base pairing could occurwithin the loop). This predicted structure is the third most stablestructure predicted by the Mfold program (Zuker (1989) Science244:48-52). A curious example of possible covariance is observed atpositions 15 and 25 in the loop region. A15 and G25 were observed tocovary to C15 and U25 in 2 clones (#18 and #29). Ligand 21 a-4 also hasthe C/U combination at the bottom of its putative loop.

[0199] Of 34 bases, 11 are “invariant” among these 25 clones (Table 16).All of the invariant positions are predicted to occur in the loop andbulge regions except C34, the last nucleotide. The last base of all 3truncated TGFβ2 ligands (FIG. 8) is a C. Removal of this C results inloss of binding. If invariant positions indicate regions where TGFβ2binds the NX22284 ligand, then binding may occur primarily in the bulgeand stem loop regions. The stems must be base paired, but can vary insequence implying that the structure of the stems may be more importantthan their sequence. The stem may be a structure used to present thebulge, loop and C34 nucleotides in the proper orientation to bind TGFβ2.

[0200] Clone 5a-11 from the biased SELEX is similar to clone 21-4 fromthe primary SELEX, particularly at positions that are invariant inclones from the biased SELEX, thus reinforcing the new structural modeland the importance of the invariant positions. It has not been possibleto fit ligand 14i-1 into a similar structure. Perhaps it represents asecond sequence motif capable of binding TGFβ2.

[0201] Binding of Nucleic Acid Ligands Isolated from the Biased SELEX

[0202] The binding of clones from the biased SELEX was compared to thebinding of full length ligand 21a-21. The majority of the clones boundas well to TGFβ2 as 21a-21 (Table 16). One clone (#20) bound about6-fold worse and one clone (#13) bound about 5-fold better than fulllength ligand 21 a-21. The average K_(d) of the clones (weighting clonesfound more than once) is 1.2 nM, which agrees with the round 5a poolK_(d) of 1 nM. Thus the ligands that were isolated in this manner werenot vastly different in affinity from the starting sequence.

[0203] One would expect there to be an optimal number of changes thatresults in higher affinity ligands. Clones with only a few changes mightbe expected to bind about the same as the starting sequence, clones witha threshold number of changes may bind better, and clones with too manychanges may bind worse. Indeed there may be a correlation between thenumber of changes and the affinity. Clones with 1 to 4 changes tend tobind the same or worse than ligand 21a-21. Clones with 5-8 changes tendto bind better than ligand 21a-21. The worst binder (#20) was the onewith the most changes (9). The ligand that bound to TGFβ2 the best(clone #13) had 7 changes relative to the starting sequence.

[0204] When the clones that bound better and those that bound worse arealigned (Table 17) it appears that an A at position 5 may be importantfor higher affinity binding since the ligands that bind to TGFβ2 bestall have an A at position 5 and all clones with an A at position 5 bindat least as well as 21a-21. In contrast clones with a U, C or G atposition 5 tend to bind worse than 21a-21. With regard to the pattern ofbase pair changes in the putative stems there is no single change thatcorrelates with better binding. In addition, the better binders do notconsistently have GC-rich stems. However the pattern of changes in thestems of the poor binders does not overlap with that seen in the stemsof the better binders. Thus, various stem sequences may result in betterbinding for subtle reasons.

[0205] A point mutant that eliminated binding of the full length 21-21transcript (21 a-21 (ML-107); Table 11) changes U at position 6 to G. AG was found at position 6 in three clones from the biased SELEX (#4, 9and 35), one of which (clone #4) has only one other base change whilethe others had additional changes. All three clones from the biasedSELEX that have a G at position 6 bind TGFβ2. Thus it would seem thatthe U6G change alone eliminates binding, but this binding defect can bereversed when combined with other sequence changes.

[0206] To summarize, some changes (such as A at position 5) may actindependently and be able to confer better binding alone, while otherschanges (e.g, at position 6 and in the stems) may influence binding in amore unpredictable way that depends on what other changes are alsopresent.

[0207] Presumably sequences that lack an “invariant” nucleotide wouldnot bind to TGFβ2. Some of the invariant bases have been deleted andothers have been changed (Table 11). None of these 10 altered sequences[21a-4(ML-111); 21a-21(ML-96, 97, 101, 102, 103, 104, 105, 120, NX22286]bind to TGFβ2.

Example 4 Substitutions of 2′-OH Purines with 2′-OCH₃ Purines in NX22284

[0208] Substitutions of 2′-OH purines with 2′-OCH₃ purines sometimesresults in nucleic acid ligands that have a longer half life in serumand in animals. Since the nucleic acid ligands described here areultimately intended for use as diagnostics, therapeutics, imaging orhistochemical reagents the maximum number of 2′-OH purines that could besubstituted with 2′-OCH₃ purines in ligand NX22284 was determined.NX22284 is a 34-mer truncate of the 70 base long 21a-21TGFβ2 ligand(Table 18). NX22284 has 17 2′-OH purines and binds about 2-fold worsethan ligand 21 a-21.

[0209] Initially an all 2′-OCH₃ purine substituted sequence wassynthesized (NX22304). Another sequence has all 2′-OH purinessubstituted with 2′-OCH₃ purines except six purines at its 5′ end.Neither bound to TGFβ2 or had measurable bioactivity (Table 18).

[0210] Therefore a set of sequences was synthesized (NX22356-NX22360;Table 18) such that groups of 3 or 4 2′-OH purines were substituted with2′-OCH₃ purines. The binding of NX22357 was reduced about 2-fold and thebioactivity was reduced 10-fold. The binding and bioactivity of NX22356,NX22258 and NX22360 were unaffected. In contrast the binding of NX22359was reduced over 100-fold and its bioactivity was reduced over 30-fold.Therefore the sequence of NX22359 was “deconvoluted” one base at a timein order to determine which individual purines in NX22359 cannot be2′-OCH₃ purines NX22374, NX22375 and NX22376 are deconvolutions ofNX22359. All three of these sequences had greatly reduced binding andbioactivity. This suggests that G20, A22, and A24 cannot be 2′-OCH₃purines.

[0211] NX22377 was designed to determine if a sequence with anintermediate number of 2′-OCH₃ purines could bind TGFβ2 and retainbioactivity. NX22377 has 10 2′-OCH₃ purines out of 17 (representing the2′-OCH₃ purines in NX22356, NX22357 and NX22360). The binding andbioactivity of NX22377 are identical to NX22284.

[0212] NX22417 was designed to test the possibility that G20, A22 andA24 must be 2′-OH purines in order to retain binding and bioactivity. InNX22417 G20, A22 and A24 are 2′-OH purines while the other 14 purinesare 2′-OCH₃. NX22417 binds to TGFβ2 as well as NX22284, but itsbioactivity is reduced about 10 fold. Since substitution of G20(NX22374) or A24 (NX22376) alone had a less severe effect thansubstitution of A22 (NX22375), nucleic acids were synthesized that hadall 2′-OCH₃ purines except position A22 (see NX22384) or G20 and A22(see NX22383). NX22383 and NX22384 did not bind or inhibit TGFβ2, againsuggesting that at least 3 purines at positions 20, 22 and 24 must be2′OH to retain binding and bioactivity.

[0213] NX22384 was analyzed by mass spectroscopy to ensure its lack ofbinding and inhibitory activity was not due to incomplete deprotectionor an incorrect sequence. The results are that NX22383 may be 0.5-0.9daltons more than the predicted molecular weight and therefore is verylikely to be what it should be.

[0214] Since NX22357 bound to TGFβ2 slightly worse than NX22284, but hada 10-fold reduced bioactivity, it was possible that one or more of thethree 2′-OCH₃ purines in NX22357 (G5, A8, A11) may also be required forbioactivity. This notion was tested by synthesizing NX22420 and NX22421.NX22421 has all three of these bases (G5, A8 and A11) as 2′-OH purines(along with G20, A22 and A24, which require 2′-OH groups). NX22420 hasA8 (along with G20, A22 and A24) as 2′-OH purines. NX22421 has G5, A8,and Al 1 (along with G20, A22 and A24) as 2′-OH purines. A8 was retainedas a 2′-OH purine in both NX22420 and NX22421 because it was invariantamong the clones from the biased SELEX and therefore it was inferredthat A8 might be less tolerant to change at the 2′ ribose position (aswas the case for G20, A22 and A24). Indeed both NX22420 and NX22421 hadapproximately the same binding and inhibitory activity as NX22284. Insummary, the NX22284 sequence can retain maximal binding and inhibitoryactivity when four purines (A8, G20, A22 and A24) are 2′-OH and theother purines are 2′-OCH₃. Note that all four of these positions wereinvariant among the clones isolated using the biased SELEX method.

[0215] While studies were being done on substituting the 2′-OH purinesof NX22284, two shorter versions of NX22284 (21a-21 [ML-130] and 21a-21[ML-134]; Table 11) were discovered that bound well to TGFβ2 astranscripts. The 2′-OCH₃ purine substitution pattern of NX22420 wastransferred to these sequences. NX22426 is the 2′-OCH₃ purine analog of21a-21(ML-134) and NX22427 is the 2′-OCH₃ purine analog of21a-21(ML-130). NX22426 bound well to TGFβ2, but had 25-fold reducedbioactivity. NX22427 may have slightly better binding and inhibitoryactivity than NX22284.

[0216] In summary, the human TGFβ2 ligand isolated by using combinedspot, spr and filter SELEX methods which have the best combination ofaffinity, short length, and inhibitory activity is NX22427, a 32-merwith 12 2′-OCH₃ purines out of a total of 16 purines.

[0217] Substitutions of 2′-OH Purines with 2′-OCH₃ Purines in NX22385

[0218] Some of the ligands that were isolated using the biased SELEXmethod (e.g., clone 13) bound better to TGFβ2.

[0219] To compare the properties of a truncated clone 13 to truncated21a-21, NX22385 was synthesized. NX22385 (Table 19) is a 34 base long,2′-F pyrimidine, 2′-OH purine version of biased SELEX clone #13. Itbinds about 2.5-fold better than NX22284, the corresponding 34 base longtruncate of 21 a-21, but its inhibitory activity is about 4-fold worse.

[0220] For reasons mentioned in the previous section it was of interestto determine if the properties of a truncated clone 13t when synthesizedas a 2′-F pyrimidine, 2′-OCH₃ purine nucleic acid. Two 2′-OCH₃ purineversions of NX22385 (NX22424 and NX22425; Table 19) were synthesizedbased on the 2′-OCH₃ pattern of NX22420, a truncate of 21a-21. In bothnucleic acids A8, G20, A22 and A24 were retained as 2′-OH purines, as inNX22420. In NX22424, the purines that are unique to clone 13 (A5, A6 andG12) are 2′-OH purines. In NX22425, those purines are 2′-OCH₃ purines.Analogs of NX22424 and NX22425 were also synthesized in which A24(NX22386) or G20 and A24 (NX22387) are 2′-OCH₃ purines. NX22386 andNX22387 were expected to serve as negative controls since 2′-OCH₃ G20 orA24 version of NX22284 were inactive. As expected NX22386 and NX22387did not bind or inhibit TGFβ2. NX22424 and NX22425 bound to TGFβ2 aswell as NX22284, but were reduced >100-fold in bioactivity (Table 19).Therefore, while other sequences that bind as well as NX22284 wereisolated, no other sequence was identified that have better bioactivity.

Example 5 Pharmacokinetic Properties of NX22323

[0221] NX22323 is a 5′-polyethylene glycol-modified version of NX22284(see Table 11; FIG. 9). The plasma concentrations of NX22323 weremeasured in rats over a 48 hour time period and are shown in FIG. 11with the corresponding pharmacokinetic parameters in Tables 20 and 21.These data demonstrate biphasic clearance of NX22323 from plasma with aninitial clearance half life (αT½) of 1 hour and a terminal clearancehalf life (βT½) of 8 hours. The volume of distribution at steady statewas approximately 140 mL/kg suggesting only minor distribution of theaptamer with the majority remaining in plasma and extracellular water.The clearance rate determined by compartmental analysis was 0.40mL/(min*kg). This value was consistent with other aptamers with similarchemical composition (5′-PEG 40K, 3′-3′ dT, 2° F. pyrimidine, 2′-OHpurine nucleic acid). These data support daily administration of NX22323for efficacy evaluation.

Example 6 2′-O-Methyl Modification of Lead Truncate Ligand CD70

[0222] TGFβ1 nucleic acid ligands are disclosed in U.S. patentapplication Ser. No. 09/275,850, filed Mar. 24, 1999, entitled“Truncation SELEX Method,” which is incorporated herein by reference. Alead aptamer was generated by truncation SELEX by hybridization (seeTable 11, Family 4, Ligand #70 in U.S. patent application Ser. No.09/275,850), herein called CD70. CD70 derivative oligonucleotides weresynthesized containing 2′-OMe modifications at various positions assummarized in Table 22 (SEQ ID NOS: 194-216). The results suggest that13 out of 16 purines can be substituted with their 2′-OMe counterpartswithout any loss of activity. The molecule with the maximum 2′-OMemodifications (CD70-m13) is also bioactive (Table 22). FIG. 10 shows aputative structure of CD70-ml 3 and the positions of that require thepresence of 2′-OH nucleotides. Of interest is the A position at the3′end of the molecule which according to the proposed structure does notparticipate in a secondary structure. Deletion of this single stranded Aaffects somewhat the binding activity of the molecule but it completelyeliminates its bioactivity (Table 22). The 2′-OH bases and the 3′ finalA are in close proximity in the proposed structure. This suggests adomain of the molecule responsible for target binding. Under thesecircumstances, it is expected that the loop shown at the top of theproposed structure (FIG. 10) may not be necessary for binding. This wasconfirmed by replacing such a loop with a PEG linker and showing thatsuch modified molecules retain binding (Table 22). The PEG linker wasconjugated to the aptamer as shown in U.S. patent application Ser. No.08/991,743, filed Dec. 16, 1997, entitled “Platelet Derived GrowthFactor (PDGF) Nucleic Acid Ligand Complexes,” which is herebyincorporated by reference in its entirety. The shortest binding aptameridentified from these experiments is CD70-m22, a 34-mer (including thePEG linker). TABLE 1 Sequences used during SELEX. (all are shown in a5′ to 3′ direction, and separated by a blank every 10 bases) Sequencesinvolved in SELEX process: (P0; DNA template for round 0 of spot SELEX)TCGGGCGAGT CGTCTGNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 (SEQ ID NO:1)NNNNNNCCGC ATCGTCCTCC C 71 A=dA; C=dC; G=dG; T=dT; N+25% each of dA, dC,dG, or dT (5′N7; primer used in PCR steps of SELEX) TAATACGACTCACTATAGGG AGGACGATGC GG 32 (SEQ ID NO:2) A=dA; C=dC; G=dG; T=dT (3′N7;primer used in RT and PCR steps of SELEX) TCGGGCGAGT CGTCTG 16 (SEQ IDNO:3) A=dA; C=dC; G=dG; T=dT (Transcription template for round 0 of spotSELEX) TAATACGACTCACTATAGGGAGGACGATGCGG-40N-CAGACGACTCGCCCGA 88 bp (SEQID NO:4) ATTATGCTGAGTGATATCCCTCCTGCTACGCC-40N-GTCTGCTGAGCGGGCT (SEQ IDNO:5) A=dA; C=dC; G=dG; T=dT; N=25% each of dA, dC, dG, or dT (R0 40N7;nucleic acid library for round 0 of spot SELEX) GGGAGGACGA UGCGGNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 (SEQ ID NO: 6) NNNNNCAGAC GACUCGCCCGA 71 A=2′-OH A; C=2′-F C; G=2′-OH G; N=25 % each of 2′-OH A, 2′-F C,2′-OH G, and 2′-F U; U=2′-F U (34N7.21a-21 DNA template for round 0 ofbiased SELEX) GGGAGGACGA TGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNC 50(SEQ ID NO:7) AGACGACTCG CCCGA 65 A=dA; C=dC; G=dG; T=dT, N=62.5 %NX22284 sequence as DNA and 12.5% of the other 4 nucleotides (dA, dC,dG, or dT) at each position (Transcription template for round 0 ofbiased SELEX) TAATACGACTCACTATAGGGAGGACGATGCGG-34N-CAGACGACTCGCCCGA 82bp (SEQ ID NO:8) ATTATGCTGAGTGATATCCCTCCTGCTACGCC-34N-GTCTGCTGAGCGGGCT(SEQ ID NO:9) A=dA; C=dC; G=dG; T=dT, N=62.5 % NX22284 sequence as DNAand 12.5% of the other 4 nucleotides (dA, dC, dG, or dT) at eachposition (34N7.21a-21 nucleic acid library for round 0, biased SELEX)GGGAGGACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNC 50 (SEQ ID NO:10)AGACGACUCG CCCGA 65 A=2′-OH A; C=2′-F C; G=2′-OH G; N=62.5 % NX22284sequence and 12.5% of other 4 nucleotides (2′-OH A, 2′-F C, 2′-OH G, or2′-F U) at each position; U=2′-F U Sequences used for subcloning,screening, sequencing ligand (ML-34; used for subcloning) CGCAGGATCCTAATACGACT CACTATA 27 (SEQ ID NO:11) A=dA; C=dC; G=dG; T=dT (ML-78; usedfor subcloning) GGCAGAATTC TCATCTACTT AGTCGGGCGA GTCGTCTG (SEQ ID NO:12)A=dA; C=dC; G=dG; T=dT (RSP1 ; vector-specific primer used to screentransformants for ligand inserts) AGCGGATAAC AATTTCACAC AGG 23 (SEQ IDNO:13) A=dA; C=dC; G=dG; T=dT (FSP2; vector-specific primer used toscreen transformants for ligand inserts) GTGCTGCAAG GCGATTAAGT TGG 23(SEQ ID NO:14) A=dA; C=dC; G=dG;T=dT (RSP2; primer for sequencingligands) ACTTTATGCT TCCGGCTCG 19 (SEQ ID NO:15) A=dA; C=dC; G=dG; T=dTSequences used to detect specific ligands (ligand 14i-1 specific primer;ML85) GCCAAATGCC GAGAGAACG 19 (SEQ ID NO:16) A=dA; C=dC; G=dG; T=dT(ligand 21a-4 specific primer; ML-79) GGGGACAAGC GGACTTAG 18 (SEQ IDNO:17) A=dA; C=dC; G=dG; T=dT (ligand 21a-21 specific primer; ML-81)GGGAGTACAG CTATACAG 18 (SEQ ID NO: 18) A=dA; C=dC; G=dG; T=dT Sequencesused for RNAse H cleavage (5′N7 cleave) CCGCaugcuc cuccc 15 (SEQ ID NO:19) a=2′-OCH₃ A; c=2′-OCH₃ C; C=dC; g=2′-OCH₃ G; G=dG; u=2′-OCH₃ U (3′N7cleave) ucgggcgagu cgTCTG 16 (SEQ ID NO:20) a=2′-OCH₃ A; c=2′-OCH₃ C;C=dC; g=2′-OCH₃ G; G=dG; u=2′-OCH₃ U; T=dT

[0223] TABLE 2 Conditions and results of filter SELEX Round^(a)[RNA]^(b), nM [TGFβ2], nM RNA^(b)/protein [Competitor] % Bound %Background Bound/Background Kd(nM)  9b  1 nM 100 nM  0.01 100 μM tRNA4.2 1.1 4 nd 10b  1 nM 30 nM 0.03 100 μM tRNA 4.3 0.13 33 100 11a  1 nM30 nM 0.03 100 μM tRNA 1.5 0.2 8 75 12d 0.2 nM  20 nM 0.01 250 μM tRNA2.2 0.3 7 40 13i 0.4 nM  10 nM 0.04 10 μM tRNA 2.6 0.16 16 30 14i 0.1nM  10 nM 0.01 10 μM heparin 14.5 0.55 20 75 15c 10 nM 10 nM 1.0 0 8.82.2 4 30 16a 55 nM 10 nM 5.5 0 9.6 2.1 5 10 17a 30 nM  3 nM 10 0 1.90.17 11 5 18b 15 nM  3 nM 5 0 2.3 0.6 4 5 19a  7 nM 0.1 nM  70 0 0.170.05 3 2 20a 0.33 nM   0.03 nM   11 0 0.1 0.04 3 1 21a 0.63 nM   0.03nM   21 0 0.3 0.1 3 1 22a 0.07 nM   0.01 nM   7 0 0.12 0.09 1 1

[0224] TABLE 3 Conditions and results of Spot SELEX Protein RNA Washes¹Signal/ Rd (pmoles) (pmoles) (μl/min) Noise % Input IncubationPre-adsorb² 1 *200 2000 2 (500/10)  4.90  ND³ 4 hrs, 20° C. No 2 *2001500 2 (1000/10) 1.80 ND 0.5 hrs, 37° C. 5 layers, 0.75 hrs 3 *200 15002 (1000/10) 5.50 ND 1 hr, 37° C. 5 layers, 1 hr 4 200 1000 2 (1000/10)11.20 0.18 1 hr, 37° C. 5 layers, 2.5 hrs *67 1000 2 (1000/10) 3.70 0.061 hr, 37° C. 5 layers, 2.5 hrs 22 1000 2 (1000/10) 1.58 0.03 1 hr, 37°C. 5 layers, 2.5 hrs 5 67 100 2 (1000/20) 26.00 1.30 1 hr, 37° C. 10layers, 0.75 hrs *22 100 2 (1000/20) 11.00 0.56 1 hr, 37° C. 10 layers,0.75 hrs 7.3 100 2 (1000/20) 2.70 0.10 1 hr, 37° C. 10 layers, 0.75 hrs6 22 50 2 (1000/20) 20.70 1.00 1 hr, 37° C. 10 layers, 0.75 hrs *7.3 502 (1000/20) 4.00 0.20 1 hr, 37° C. 10 layers, 0.75 hrs 2.4 50 2(1000/20) 1.20 0.06 1 hr, 37° C. 10 layers, 0.75 hrs 7 22 7 3 (1000/50)24.00 1.30 1 hr, 37° C. 10 layers, 1.5 hrs *7.3 7 3 (1000/50) 7.50 0.401 hr, 37° C. 10 layers, 1.5 hrs 2.4 7 3 (1000/50) 1.50 0.07 1 hr, 37° C.10 layers, 1.5 hrs 8 *7.3 3 2 (1000/60) 77.00 0.41 0.75 hr, 37° C. 10layers, 1.5 hrs 2.4 3 2 (1000/60) 8.50 0.04 0.75 hr, 37° C. 10 layers,1.5 hrs 0.7 3 2 (1000/60) 1.00 ND 0.75 hr, 37° C. 10 layers, 1.5 hrs 9*7.3 1 2 (1000/20) 87.00 0.23 1 hr, 37° C. 10 layers, 1.5 hrs 2.4 1 2(1000/20) 4.00 0.01 1 hr, 37° C. 10 layers, 1.5 hrs 0.7 1 2 (1000/20)2.50 0.006 1 hr, 37° C. 10 layers, 1.5 hrs 10 7.3 <1 (no tRNA) 2(1000/20) 13.70 ND 0.5 hr, 37° C. 10 layers, 1.5 hrs 7.3 <1 (10¹ tRNA)⁴2 (1000/20) 10.50 ND 0.5 hr, 37° C. 10 layers, 1.5 hrs 7.3 <1 (10² tRNA)2 (1000/20) 5.00 ND 0.5 hr, 37° C. 10 layers, 1.5 hrs 7.3 <1 (10³ tRNA)2 (1000/20) 1.80 ND 0.5 hr, 37° C. 10 layers, 1.5 hrs

[0225] TABLE 4 Conditions and results surface plasmon resonancebiosensor (spr) SELEX. Progress of BIA SELEX with TGFβ2 TGFβ2, RU¹[RNA], Injections Fractions Fraction RU after Rd FC1 FC2 FC3 FC4 μM²(vol, μL)³ (min each)⁴ FW⁵ SDS⁶ 2 1293 874 294 0 4 4 (40) 3 (5) 3rd &SDS ˜100  3 1176 1178 1181 0 15 4 (40) 3 (5) 3rd & SDS ˜50-100 4 30102037 1767 0 10 6 (40) 3 (5) 3rd & SDS ˜80 5 5520 5334 4265 0 5 6 (40) 3(5) 3rd & SDS ˜100-150  6 4075 3143 298 0 5 6 (40) 3 (5) 3rd & SDS˜75-100 7 3773 2616 2364 0 2 6 (40) 3 (5) 3rd & SDS ˜330-220  8 25741842 1461 0 5 4 (40) 3 (5) 3rd & SDS ˜60-105 9 3180 2029 1688 0 3 4 (40)3 (5) 3rd & SDS ˜77-114 10  344 718 1692 0 1 4 (40)  6 (10) 6th & SDS˜50 11  217 675 386 0 5 2 (40)  6 (10) 6th & SDS ˜50-62 

[0226] TABLE 5 Sequences isolated from round 8 of surface plasmonresonance SELEX. NAME^(a) SEQ ID NO. SEQUENCE^(b) BINDING^(c) 8.1 (1) 21GGGAGGACGAUGCGG UCCUCAAUG-AUCUU---------UCCUGUUUAUGCUCCCCAGACGACUCGCCCGA FILTER 8.2 (1) 22 GGGAGGACGAUGCGG AAGUAACGUUUAAGUAAAAUUCGUUCUCUCGGUAUUUGGC CAGACGACUCGCCCGA TGFβ2 8.3 (14) 23GGGAGGACGAUGCGG AAGUAACGUUGAAGUAAAAUUCGUUCUCUCGGCAUUUGGCCAGACGACUCGCCCGA TGFβ2 8.5 (1) 24 GGGAGGACGAUGCGGUCCUAACCAUCACAAUCUCAAUUCUUAUAUUUUCCCGCCC CAGACGACUCGCCCGA NONE 8.6 (1)25 GGGAGGACGAUGCGG --AAACCAAAAGACCACAUCUCCAUACUCACGCUCUGCCCCAGACGACUCGCCCGA NONE 8.8 (1) 26 GGGAGGACGAUGCGGAUAGAUCGGUCCGAUAAGUCUUUCAUCUUUACCUGGCCCC CAGACGACUCGCCCGA NONE 8.9 (4)27 GGGAGGACGAUGCGG AAGUAACGUUGAAGUAAAAUUCGUUCUCUCGGUAUUUGGCCAGACGACUCGCCCGA TGFβ2 8.11 (1) 28 GGGAGGACGAUGCGGACGAUCCUUUCCUUAACAUUUCAUCAUUUCUCCUGUGCCC CAGACGACUCGCCCGG FILTER 8.12(1) 29 GGGAGGACGAUGCGG UCCAUCAACAAUCUUAUCAUUAUGUUUUUCCUUCCCGCCCCAGACGACUCGCCCGA NONE 8.13 (1) 30 GGGAGGACGAUGCGGUCCUCUGAGCCGAUCUUCUUCACUACUUCUUUUUCUGCCC CAGACGACUCGCCCGA FILTER 8.15(2) 31 GGGAGGACGAUGCGG UUCCUCAAUUCUUCCAUCUUCAUAAUGUUUCCCUUUGCCCCAGACGACUCGCCCGA FILTER 8.18 (1) 32 GGGAGGACGAUGCGGUCUACCCUUUAGCAGUAUUUGUUUCCAUCGUUGUUUGCCC CAGACGACUCGCCCGG NONE 8.20 (1)33 GGGAGGACGAUGCGG UCUCAACGAAGAACAUCGUUGGAUACUGUUUGUCCCGCCCCAGACGACUCGCCCGA NONE 8.21 (1) 34 GGGAGGACGAUGCGGUUCAGUUUCCUUCAGUUUUCGUUUCUAAUUCUUGUGUCCC CAGACGACUCGCCCGA FILTER 8.22(1) 35 GGGAGGACGAUGCGG ----------AGCGGAUUAAUUAGUCUGACUUCUUGUCCCCAGACGACUCGCCCGA 8.23 (1) 36 GGGAGGACGAUGCGGAGACAUCUUUGUCUCGAUUAGUCAUGUUCCUUACCUGCCC CAGACGACUCGCCCGA NONE 8.24 (1)37 GGGAGGACGAUGCGG --UCCUCUAGCAAGCAGCUUCUCAUCUUAUUUUUCCGCCCCAGACGACUCGCCCGA 8.25 (1) 38 GGGAGGACGAUGCGGUGCACAGUGAUGGAUGACAUUGUAUAACGGUAUGCGUCCC CAGACGACUCGCCCGA 8.26 (1) 39GGGAGGACGAUGCGG -ACCUAUCUUUCUUCCAAGUCAUAGUUUUACUUCCCGCCCCAGACGACUCGCCCGA FILTER 8.28 (1) 40 GGGAGGACGAUGCGGAUGACACCUAAUCAUCGAUCCGCUAUCUAAAACCUCACCC CAGACGACUCGCCCGA NONE 8.29 (1)41 GGGAGGACGAUGCGG UCCUCAGACAAAUCUUUCUUGAAUCUUUCCUUAACUGCCCCAGACGACUCGCCCGA FILTER 8.31 (1) 42 GGGAGGACGAUGCGG-ACCGAUUCUCCAACUUGACAUUUAUUCCUCUUUCUGCCC CAGACGACUCGCCCGA FILTER 8.33(1) 43 GGGAGGACGAUGCGG UCCUCUGAGCCAAUCUUCUUCGCUACUUCUUUUUCUGCCCCAGACGACUCGCCCGA FILTER 8.34 (1) 44 GGGAGGACGAUGCGGAUUCUUUCUCCAACGCUUUUCACUACCUACAUUUCUGCCC CAGACGACUCGCCCGA FILTER 8.35(1) 45 GGGAGGACGAUGCGG AUCCUAUCCUCUGAAUAUCAUUAAAUCAUCUUCUCCGCCCCAGACGACUCGCCCGA NONE 8.36 (1) 46 GGGAGGACGAUGCGGUUCAAUCAUCUUCACUCU-CAUUUCCUUUUUCCUACUCCC CAGACGACUCGCCCGA FILTER 8.38(1) 47 GGGAGGACGAUGCGG CGAUAGAAUCUAGUCGUUCUAGAUGAUCUGGUACGUGCCCCAGACGACUCGCCCGA 8.39 (1) 48 GGGAGGACGAUGCGGUAGUAAUCCUUGUCUUCCAUUUCUCUUUACCCUUUUGCCC CAGACGACUCGCCCGA FILTER 8.40(1) 49 GGGAGGACGAUGCGG ----CCCAUUAGUCCUCAUUAGU------CCCCUGUGCCCCAGACGACUCGCCCGA NONE 8.41 (1) 50 GGGAGGACGAUGCGGCAUCUUAUCCUCCAUCAGUUACUCUUCGUUAUUCCCGCCC CAGACGACUCGCCCGA 8.45 (1) 51GGGAGGACGAUGCGG UCC-AAAUCCUCUUCCCAUGUUAGCAUUCAGCCUUGUCCCCAGACGACUCGCCCGA 8.46 (1) 52 GGGAGGACGAUGCGG-UUCCGACAAUUUCCUCCACCAUUAGAUUUCUUGCUGCCC CAGACGACUCGCCCGA 8.47 (1) 53GGGAGGACGAUGCGG UCUUGAUCCUCCUUUGUGUCUUUCUUUGUCUUCCCUGCCCCAGACGACUCGCCCGA 8.48 (2) 54 GGGAGGACGAUGCGGAAGUAACGUUGAAGUAAAAUUCGUUCUCUCGGUAUU-GGC CAGACGACUCGCCCGA TGFβ2 8.49 (1)55 GGGAGCACGAUGCGG -UCCGAUCAGUUCCUUCGAUUAAUCUUCUUUCCUGCCCCCCAGACGACUCGCCCGA 8.51 (1) 56 GGGAGGACGAUGCGGAAUCCUUCUCCCUGAUGAAUAUGACCUUUUUCUUGCUCCC CAGACGACUCGCCCGA 8.52 (1) 57GGGAGGACGAUGCGG AUGAUCUUUAAUGUCUGGUUUGAGGUCAAUGCGGGUGCCCCAGACGACUCGCCCGA 8.56 (1) 58 GGGAGGACGAUGCGGAGAUGGUACUCCAUCUCCUUUAUGUGCCCAUCGCUGUCCC CAGACGACUCGCCCGA 8.57 (1) 59GGGAGGACGAUGCGG UCCUC-GAUUCU---------AAUUUACUCCUUUUUCCCCCAGACGACUCGCCCGA 8.61 (1) 60 GGGAGGACGAUGCGGUCUACCCUUUAGCAGUAUUUGUUUCCAUCGUUGUUUGCCC CAGACGACUCGCCCGA 8.62 (1) 61GGGAGGACGAUGCGG -CACAAUAUUCUCCUCUACUUCCACGUAUUUUCCUGUCCCCAGACGACUCGCCCGA 8.64 (1) 62 GGGAGGACGAUGCGGUCCUCAACCUUAGACUUUCAUUUCUUCAGUUCUUCUGCCC CAGACGACUCGCCCGA 8.65 (1) 63GGGAGGACGAUGCGG UAGUGGUCUGUCAAAGGAAUAGCUAGUAGUGUUUGGUCCCCAGACGACUCGCCCGA 8.69 (1) 64 GGGAGGACGAUGCGGCAUCUUCCUUAGCAUACCAGUUUAUUCCUUUCCCUGUCCC CAGACGACUCGCCCGA 8.71 (1) 65GGGAGGACGAUGCGG AGCGACAGUAUAGUUAGUACUCUAGCUCUAGUGCUGUCCCCAGACGACUCGCCCGA 8.72 (1) 66 GGGAGGACGAUGCGGACCUCUCAUGAUCAGCAUCUCGCGUAAUCACGGUUCACCC CAGACGACUCGCCCGA 8.74 (1) 67GGGAGGACGAUGCGG UCCGUACUCCAUUUCCUAUUUGAUUCCUUUUCCUCUGCCCCAGACGACUCGCCCGA 8.75 (1) 68 GGGAGGACGAUGCGGAACCCACGACCUUACCUUAAUCAUGUAUUUCUCUCUGCCC CAGACGACUCGCCCGA 8.76 (1) 69GGGAGGACGAUGCGG ------AGAUAAUGAGUGACGGUGAUUAUAGAUGCUGCCCCAGACGACUCGCCCGA 8.79 (1) 70 GGGAGGACGAUGCGGUUCCUCAAUUCUUCCAUCUUCAUAAUGUUUCCCUUUGCCC CAGACGACUCGCCCGA 8.80 (1) 71GGGAGGACGAUGCGG UUCCU-------UCCAACGUUAUCUACUUUCU----GCCCCACACGACUCGCCCGA

[0227] TABLE 6 Conditions and results of resonant mirror (rm) opticalbiosensor SELEX. Progress of IASYS SELEX with TGFβ2 TGFβ2, Arcsec¹[RNA], Binding Dissociation Rd C1 C2 μM² Vol, μL³ (min)⁴ (min)⁵ Elution⁶10 1777 0 1 50 27 29 water 11 1777 0 10 50 30 60 water 12 1777 0 10 5060 150 water 13 1893 0 0.05 50 37 73 water & SDS 14 1721 0 3.5 50 30 35water & SDS

[0228] TABLE 7 Sequences isolated from round 13 of resonant mirror SELEXNAME^(a) SEQ ID NO. SEQUENCE^(b) 14i-1 72 GGGAGGACGAUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGG-CAUUUGGC CAGACGACU-CGCCCGA 13.20 (1) 73GGGAGGACGAUGCGG AAGUAACGUUAUAGUAAAAUUCGUUCUCUCGG-UAUU_GGCCAGACGACU-CGCCCGA 13.22 (2) 74 GGGAGGACGGUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGG-CGUUUGGC CAGACGACU-CGCCCGA 13.24 (2) 75GGGAGGACGAUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGG-CGUUUGGUCAGACGACU-CGCCCGA 13.30 (1) 76GGGAG_ACGAUGCGG AAGUAACGUUGUAGUAAAAUUCGUUCUCUCGG-CAUUUGGCCAGACGACU-CGCCCGA 13.32 (1) 77 GGGAGGACGAUGCGGAAGUAACGUUGAAGUAAAAUUCGUUCUCUCUG-CGUUUGGUCAGACGACU-CGCCCGA 13.34 (1) 78GGGAGGACGAUGCGG AAGUAACGUUGAAGUAAAAUUCGUUCUCCUGG-UA_UUGGCCAGACGACU-CGCCCGA 13.36 (2) 79 GGGAGGACGAUGCGGAAGUAACGUUGAAGUAAAAUUCGUUCUCUCGG-CAUUUGGC CAGACGACU-CGCCCGA 13.40 (1) 80GGGAGGACGAUGCGG AAGUAACGUUGUAGUAAAAUUCGUUCUCUUGG-CAUUU_GCCAGACGACU-CGCCCGA 13.42 (1) 81 GGGAGGACGAUGCGGAAGUAACGUUAAAGUAAAAUUCGUUCUCUCGG-CGUUUGGC CAGACGACU-CGCCCGA 13.44 (1) 82GGGAGGACGAUGCGG AAGUAACGUUGAAGUAAAAUUCGUUCUCUCGG-CGUUUGGCCAGACGACU-CGCCCGA 13.48 (1) 83 GGGAGGACGAUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGG-UAUUUGGC CAGACGACU-CGCCCGA 13.50 (1) 84GGGAGGACGAUGCGG AAGUAACGUUGUAGUAAAAUUCGUUCUCUUGG-UCUU_GGCCAGACGACU-CGCCCGA 13.54 (1) 85_GGAGGACGAUGCG_AAGUAACGUUGUAGUAAAAUUCGUUCUCUCGGGCAUUUGG_CAGACGACUUCGCCCGA

[0229] TABLE 8 Sequences and boundaries of TGFβ2 ligands isolated fromrounds 14 and 21 of filter SELEX. NAME^(a) SEQ ID NO. SEQUENCE^(b) Kd(nM) Ki (nM) 14i-1 72GGGAGGACGAUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGGCAUUUGGCCAGACGACUCGCCCGA10 230 21a-4 86 GGGAGGACGAUGCGGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGCUUGUCCCCAGACGACUCGCCCGA 3 30 21a-21 87GGGAGGACGAUGCGG-UUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCC AGACGACUCGCCCGA1 10 region:     5′ fixed                    selected                  3′fixed

[0230] TABLE 9 Number of sequences isolated using the SELEX process.SELEX round Sequence 8-spr 13-rm 14i 16a 18b 21a TOTAL 14i-1 0 0 75 2 00 77 14i-1 variants 21 15 22 2 0 0 60 21a-4 0 0 0 0 0 3 3 21a-4 variants0 0 4 7 0 2 13 21a-21 0 0 0 1 11 38 50 21a-21 variants 0 0 0 2 4 4 10unidentified 36 0 0 0 0 0 36 filter-binding 12 0 1 1 0 1 15 TOTAL 69 15102 15 15 48 264

[0231] TABLE 10 Characteristics of nucleic acid pools isolated using theSELEX method. Round^(a) Sequence of pool^(b) % of pool^(c) % oftransformants^(d) % of clones^(e) 0 random 14i-1: <0.03 6-spr random14i-1: ˜1 8-spr slightly nonrandom 14i-1: ˜5 14i-1: 30 other: 70 9-sprnonrandom 9-rm can read sequence of ligand 14i-1 10-rm can read sequenceof ligand 14i-1 11-rm can read sequence of ligand 14i-1 12-rm can readvariants of ligand 14i-1 sequence 13-rm can read variants of ligand14i-1 sequence 14i-1: 10-100 14i-1: 100 21a-21: <0.1 14i 14i-1: 9321a-4: 4 21a-21: 0.2-0.5 21a-21: 0 other: 3 16a 14i-1: 27 21a-4: 4721a-21: 3-100 21a-21: 20 other: 6 18b 21a-21: 3-100 21a-21: 100 21a21a-4: 9 21a-4: 10 21a-21: 3-100 21a-21: 90 21a-21: 84 other: 1 other: 6

[0232] TABLE 11 Truncates of human TGFβ2 nucleic acid ligand 21a-21. BIOAC- SEQ TI- ID VI- NAME SEQUENCE^(a) NO: BINDING^(b) LENGTH^(c) TY^(d)21a-21 GGGAGGACGAUGCGGUUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCCAGACGACUCGCCCGA 87 0.5 70 1 21a-21 GGGAGGACGAUGCGGUUCAGGAGGGUAUUACAGAGUCUGUAUAGCUGUACUCCC CAGACGACUCGCCCGA 88 250 34 (U6G) 21a-             GGUUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCCAGACGACUCGCCCGA89 0.5 56 21Δ5′ 21a-GGGAGGACGAUGCGGUUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCCA 90 100 56 21Δ3′21a-              GGUUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCCA 91 0.5 421 21Δ5′, 3′ 21a-21                   GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCC 92 0.5 36(ML-94) 21a-21                    GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCC 931 34 (ML-95) 21a-21                    GGAGGUUAUUACAGAGUCUGUAUAGCUGUA 941000 30 (ML-96) 21a-21                    GGAGGUUAUUACAGAGUCUGUAUAGC 951000 26 (ML-97) 21a-21                   GGAGGUUAUUACAGAGUCUGUAUAGC   CUCC 96 1000 30 (ML-99)21a-21                    GGAGGUUAUU  AGAGUCU  AUAGCUGUACUCC 97 1000 30(ML-101) 21a-21                    GGAGGUUAUU  AGAGUCU  AUAGC    CUCC 981000 26 (ML-102) 21a-21                   GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUC 99 50 33 (ML-103)21a-21                    GGAGGUUAUUACAGAGUCUCUAUAGCUGUACUC 100 70 32(ML-104) 21a-21                    GGAGGUUAUUACAGAGUCUGUAUAGCUGUAC 1011000 31 (ML-105) 21a-21                    GGAGGUUAUUACAGAGUCUGUAUAGCGUACUCC 102 1000 33 (ML-114) 21a-21                   GGAGGUUAUUACAGAGUCUGUAUAGCUCU CUCC 103 1000 33(ML-115) 21a-21                    GGAGGUUAUUACAGAGUCUGUAUAGCU  ACUCC104 1000 32 (ML-116) 21a-21                    GGAGGUUAUACAGAGUCUGUAUAGCUGUACUCC 105 1000 33 (ML-118) 21a-21                   GGAGGUUAUUACAGA UCUGUAUAGCUGUACUCC 106 1000 33(ML-120) 21a-21                    GGAGGUUAUUACA AGU UGUAUAGCUGUACUCC107 1000 32 (ML-122) 21a-21                    GGAGGUUAUUACAGAGUUGUAUAGCUGUACUCC 108 1000 33 (ML-128) 21a-21                    GGGGUUAUUACAGAGUCUGUAUAGCUGUAC CC 109 2 32 (ML-130) 21a-21                   GGAGGUUAUUAC GAGUCUGUAUAGC GUACUCC 110 1000 32(ML-132) 21a-21                    GGAGA UAUUACAGAGUCUGUAUAGCUGUACUCC111 10 33 (ML-134) 21a-21                    GG GGUUAUUCAGAGUCUGUAUAGCUG AC CC 112 10000 30 (ML-136) 21a-21                   GG GGUUAUUA AGAGUCUGUAUAGCU UAC CC 113 10000 30(ML-138) NX22283                   GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCC[3′T] 114 0.6 360.5 NX22284                    GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCC  [3′T]115 1 34 1 NX22285                   GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCCA 116 2 37NX22286                    GGAGGUUAUUACAGAGUCUGUAUAGCUGUA 117 130 30 >20NX22301                     GAGGUUAUUACAGAGUCUGUAUAGCUGUACUCC  [3′T] 1181 33 2 NX22302                     AGGUUAUUACAGAGUCUGUAUAGCUGUACUCC  [3′T] 119 100 32NX22303                       GGUUAUUACAGAGUCUGUAUAGCUGUACUCC  [3′T]120 >100 31 >100 NX22323               PEG-GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCC   [3′T] 121 nt 343

[0233] TABLE 12 Alignment of human transforming growth factor β aminoacid sequences. SEQ ID NO. TGFβ1: ALDTNYCFSS TEKNCCVRQL YIDFRKDLGWKWIHEPKGYH ANFCLGPCPY IWSLDTQYSK 60 122 TGFβ2: ALDAAYCFRN VQDNCCLRPLYIDFKRDLGW KWIHEPKGYN ANFCAGACPY LWSSDTQHSR 60 123 TGFβ3: ALDTNYCFRNLEENCCVRPL YIDFRQDLGW KWVHEPKCYY ANFCSGPCPY LRSADTTHST 60 124 TGFβ2specific:    AA      VQD   L        KR              N     AA       S     R TGFβ1: VLALYNQHNP GASAAPCCVP QALEFLPIVY YVGRKPKVEQLSNMIVRSCK CS 112 125 TGFβ2: VLSLYNTINP EASASPCCVS QDLEPLTILY YIGKTPKIEQLSNNIVKSCK CS 112 126 TGFβ3: VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQLSNMVVKSCK CS 112 127 TGFβ2 specific:  S    I            S             I K   I

[0234] TABLE 13 Truncates of human TGFβ2 nucleic acid ligand 14i-1. SECID BIND- NAME SEQUENCE^(a) NO. ING^(b) LENGTH^(c) 14i-1GGGAGGACGAUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGGCAUUUGGCCAGACGACUCGCCCGA72 1 71 14i-1Δ5′^(d)             GGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGGCAUUUGGCCAGACGACUCGCCCGA128 >100 56 14i-1Δ3′^(d)GGGAGGACGAUGCGGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGGCAUUUGGCCA 129 3 5714i-1Δ5,3′^(d)              GGAAGUAACGUUGUAGUAAAAUUCGUUCUCUCGGCAUUUGGCCA130 >100 42 14i-1t5-41    gGGAgGAUGCGGAAGUAACGUUGUAGUAAAAUUCcUUC 131 138 14i-1t5-38    gGGAgGAUGCGGAAGUAACGUUGUAGUAAAAUUCc 132 >100 3514i-1t5-35    gGGAgGAUGCGGAAGUAACGUUGUAGUAAAAU 133 >100 32 14i-1 (ML-86)   gGGAgGAUGCGGAAGuAAcGUUGUAGU     UCcUUC 134 >100 33 14i-1 (ML-87)   gGGAgGAUGCGGAAGUAACGUUGUAGU 135 >100 27 14i-1 (ML-89)          gGgaGgAGUAACGUUGUAGU 136 >100 20

[0235] TABLE 14 Truncates of human TGFβ2 nucleic acid ligand 21a-4.Bind- Name Sequence^(a) SEQ ID NO. ing^(b) Length^(c) 21a-4GGGAGGACGAUGCGGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGCUUGUCCC CAGACGACUCGCCCGA86 1 71 21a-4Δ5′^(d)             GGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGCUUGUCCCCAGACGACUCGCCCGA137 >100 56 21a-4Δ3′^(d)GGGAGGACGAUGCGGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGCUUGUCCCCA 138 1 5721a-4Δ5′,3′^(d)             GGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGCUUGUCCCCA 139 >100 4221a-4 (ML-91)       ggGgaGCGGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGCUU 140 144 21a-4 (ML-92)       ggGgaGCGGCGUUGUU       gaaa        AGUCCGCUU141 >100 27 21a-4 (ML-108)      ggGgaGCGGCGUUCUUU   CGUAUGUAUAU   AAGUCCGCUU 142 >100 38 21a-4(ML-109)       ggGgaGCGGCGUUGUUU      AUGUAU     AAGUCCGCUU 143 >100 3321a-4 (ML-110)       ggGgaGCGGCGUUGUUUAGUCGUAUGUAUAUACUAAGUCCGC 144 1 4221a-4 (ML-111)       ggGgaGCGGCGUUGUUUAGUCGUAUGUAUAUACUAAGU 145 30 38

[0236] TABLE 15 Biased SELEX conditions and results. Round^(a)[RNA]^(b), nM [TGFβ2], nM RNA^(b)/protein [Competitor] % Bound %Background Bound/background Kd (nM)^(c) 34N7.21a-21 round 0 nucleic acid870 1a 1000 150 7 0 1.4 1.4 1.0 395 2a 450 300 1.5 0 1.7 1.0 1.7 186 3a10 50 0.2 0 17.5 1.0 17.5 25 4a 50 10 5 0 11.0 0.9 12.3 17 4b 50 10 5333 nM NX22284 2.2 1.3 1.7 8 5a 8 1 8 0 1.4 0.9 1.5 1 5b 8 1 8 100 nMNX22284 0.8 0.7 1.1 17 6a 4 0.5 8 0 2.9 2.9 1.0 1 6b 6 0.5 12 100 nMNX22284 1.8 1.3 1.4 1 7a 5 0.25 20 0 0.5 0.14 3.4 1 7b 5 0.25 20 200 nMNX22284 0.15 0.1 1.5 0.5 5 mM tRNA 8a 1 0.05 20 0 1.05 1.1 0.9 1 8b 10.05 20 100 nM NX22284 0.6 0.5 1.2 3 5 mM tRNA 9a 125 1 125 0 0.6 0.51.2 nd 9b 0.9 0.01 90 0 0.15 0.14 1.0 nd

[0237] TABLE 16 Nucleic acid ligands isolated from round 5a of a humanTGFβ2 biased SELEX. SEQ ID CHAN- BIND- NAME^(a) 5′ FIXED SELECTED^(b)3′ FIXED NO: GES^(c) ING^(d) putative  S1   B    S2     L        S2  S1structural element: 21a-21:GGGAGGACGAUGCGGUUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUA CUCCC CAGACGACUCGCCCGA72 0 1.0 1: (2) GGGAGGACGAUGCGG GGUGAUUAUUACAGAGUAUGUAUAGCUGUACCCCCAGACGACUCGCCCGA 146 4 0.8 2: (1) GGGAGGACGAUGCGGAGGCGUUAUUAGAGAGUCUGUAUAGCUCUAGCCC CAGACGACUCGCC-GA 147 7 0.6 4: (1)GGGAGGACGAUGCGG GGAGGGUAUUACAGAGUAUGUAUAGCUGUACUCC CAGACGACUCGCCCGA 1482 1.4 6: (2) GGGAGGACGAUGCGG GGAGGUUAUUAUAGAGUCUGUAUAGCUAUACCCCCAGACGACUCGCCCGA 149 3 1.6 7: (1) GGGAGGACGAUGCGGGAGGGUUAUUAUAGAGUCUGCAUAGCUAUACCCC CAGACGACUCGCCCGA 150 5 0.3 9: (1)GGGAGGACGAUGCGG UGAGAGUAUUACGGAGUAUGUAUAGCCGUACCCC CAGACGACUCGCCCGA 1517 0.3 10: (1) GGGAGGACGAUGCGG GGGCAUUAUUUCAGAGUCUGUAUAGCUGUAGCCCCAGACGACUCGCCCGA 152 6 0.3 11: (2) GGGAGGACGAUGCGGGCGGAUUAUCACAGAGUAUGUAUAGCUGUGCCGC CAGACGACUCGCCCGA 153 8 0.4 13: (1)GGGAGGACGAUGCGG UGUGAAUAUUAGAGAGUCUGUAUAGCUCUACCCC CAGACGACUCGCCCGA 1547 0.2 14: (1) GGGAGGACGAUGCGG CGGGAUUAUUACUGAGUCUGUAUAGCAGUACCCCCAGACGACUCGCCCGA 155 6 0.4 15: (1) GGGAGGACGAUGCGGGUGGAAUAUUACGGAGUCUGUAUAGCCGUACUCC CAGACGACUCGCCCGA 156 6 0.4 17: (1)GGGAGGACGAUGCGG GGGGACUAUUAGUGAGUCUGUAUAGCACUACCCC CAGACGACUCGCCCGA 1578 0.8 18: (1) GGGAGGACGAUGCGG GUGGAUUAUUACAGCGUCUGUAUAUCUGUACCCCCAGACGACUCGCCCGA 158 6 1.0 19: (2) GGGAGGACGAUGCGGGCAGGUUAUUACAGAGUCUGUAUAGCUGUACUGC CAGACGACUCGCCCGA 159 2 1.0 20: (1)GGGAGGACGAUGCGG GGUAGAUAUCACUGAGUCUGUAUAGCAGUGUCCC CAGACGACUCGCCCGA 1609 5.7 21: (2) GGGAGGACGAUGCGG AGGGAUUAUUACAGAGUCUGUAUAGCUGUACCCCCAGACGACUCGCCCGA 161 4 0.7 22: (4) GGGAGGACGAUGCGGGUGGAUUAUUACAGAGUCUGUAUAGCUGUACCCC CAGACGACUCGCCCGA 162 4 1.1 25: (1)GGGAGGACGAUGCGG GGGCGUUAUUACAGAGUCUGUAUAGCUGUAGCCC CAGACGACUCGCCCGA 1634 1.0 26: (1) GGGAGGACGAUGCGG GGUGGUUAUUACACAGUAUGUAUAGGUGUACCCCCAGACGACUCGCCCGA 164 4 3.1 28: (1) GGGAGGACGAUGCGGAGGGAAUAUUACAGAGUAUGUAUAGCUGUACCCC CAGACGACUCGCCCGA 165 6 1.0 29: (1)GGGAGGACGAUGCGG GGAGUUUAUUACAGCGUCUGUAUAUCUGUAGCCC CAGACGACUCGCCCGA 1665 1.0 30: (1) GGGAGGACGAUGCGG UGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCCCAGACGACUCGCCCGA 167 1 2.4 34: (1) GGGAGGACGAUGCGGGGUGGUUAUUAGAGAGUCUGUAUAGCUCUACGCC CAGACGACUCGCCCGA 168 4 1.7 35: (1)GGGAGGACGAUGCGG GGGGAGUAUUAAAGAGUCUGUAUAGCUUUACCCC CAGACGACUCGCCCGA 1696 0.8 36: (1) GGGAGGACGAUGCGG GGAGGAUAUUAUAGAGUCUGUAUAGCUAUACCCCCAGACGACUCGCCCGA 170 4 1.9 invariant       UAU      GU UG AUA         C

[0238] TABLE 17 Highest and lowest affinity TGFβ2 nucleic acid ligandsfrom biased SELEX. NAME 5′ FIXED SELECTED^(a) 3′ FIXED SEQ ID NO.BINDING^(b) CHANGES^(c) HIGHEST AFFINITY LIGANDS: 13: GGGAGGACGAUGCGGUGUGAAUAUUAGAGAGUCUGUAUAGCUCUACCCC CAGACGACUCGCCCGA 154 0.2 7 14:GGGAGGACGAUGCGG CGGGAUUAUUACUGAGUCUGUAUAGCAGUACCCC CAGACGACUCGCCCGA 1550.4 6 21: GGGAGGACGAUGCGG AGGGAUUAUUACAGAGUCUGUAUAGCUGUACCCCCAGACGACUCGCCCGA 161 0.7 4 35: GGGAGGACGAUGCGGGGGGAGUAUUAAAGAGUCUGUAUAGCUUUACCCC CAGACGACUCGCCCGA 169 0.8 6 putativestructural elements:  S1   B    S2     L        S2  S1 21a-21:GGGAGGACGAUGCGGUUCAGGAGGUUAUUACAGAGUCUGUAUAGCUGUA CUCCC CAGACGACUCGCCCGA72 1.0 0 LOWEST AFFINITY LIGANDS: 36: GGGAGGACGAUGCGGGGAGGAUAUUAUAGAGUCUGUAUAGCUAUACCCC CAGACGACUCGCCCGA 170 2.0 4 30:GGGAGGACGAUGCGG UGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCC CAGACGACUCGCCCGA 1672.4 1 26: GGGAGGACGAUCCGG GGUGGUUAUUACACAGUAUGUAUAGGUGUACCCCCAGACGACUCGCCCGA 164 3.1 4 6: GGGAGGACGAUGCGGGGAGGUUAUUAUAGAGUCUGUAUAGCUAUACCCC CAGACGACUCGCCCGA 149 3.3 3 20:GGGAGGACGAUGCGG GGUAGAUAUCACUGAGUCUGUAUAGCAGUGUCCC CAGACGACUCGCCCGA 1605.7 9 invariant:       UAU      GU UG AUA         C

[0239] TABLE 18 Substitution of 2′-OH purines with 2′-OCH₃ purines inNX22284 ligand. NAME SEQUENCE^(a) SEQ ID NO. BINDING^(b) LENGTH^(c)BIOACTIVITY^(d) NX22284 GGAGGUUAUUACAGAGUCUGUAUAGCUGUACUCC[3′T] 115 1 341 NX22304 ggaggUUaUUaCagagUCUgUaUagCUgUaCUCC[3′T] 171 >100 34 >100NX22355 GGAGGUUAUUaCagagUCUgUaUagCUgUacUCC[3′T] 172 >100 34 >100 NX22356ggagGUUAUUACAGAGUCUGUAUAGCUGUACUCC[3′T] 173 1 34 1 NX22357GGAGgUUaUUaCAGAGUCUGUAUAGCUGUACUCC[3′T] 174 2 34 10 NX22358GGAGGUUAUUACagagUCUGUAUAGCUGUACUCC[3′T] 175 1 34 1 NX22359GGAGGUUAUUACAGAGUCUgUaUaGCUGUACUCC[3′T] 176 >100 34 >30 NX22360GGAGGUUAUUACAGAGUCUGUAUAgCUgUaCUCC[3′T] 177 1 34 1 NX22374GGAGGUUAUUACAGAGUCUgUAUAGCUGUACUCC[3′T] 178 25 34 >100 NX22375GGAGGUUAUUACAGAGUCUGUaUAGCUGUACUCC[3′T] 179 >100 34 >300 NX22376GGAGGUUAUUACAGAGUCUGUAUaGCUGUACUCC[3′T] 180 50 34 >100 NX22377ggaggUUaUUaCAGAGUCUGUAUAgCUgUaCUCC[3′T] 181 1 34 1 NX22383ggaggUUaUUaCagagUCUGUAUagCUgUaCUCC[3′T] 182 500 34 >100 NX22384ggaggUUaUUaCagagUCUgUAUagCUgUaCUCC[3′T] 183 10000 34 >100 NX22417ggaggUUaUUaCagagUCUGUAUAgCUgUaCUCC[3′T] 184 1 34 10 NX22420ggaggUUAUUaCagagUCUGUAUAgCUgUaCUCC[3′T] 185 1 34 1 NX22421ggagGUUAUUACagagUCUGUAUAgCUgUaCUCC[3′T] 186 2 34 1 NX22426ggaga-UAUUaCagagUCUGUAUAgCUgUaCUCC[3′T] 187 1 33 25 NX22427gg-ggUUAUUaCagagUCUGUAUAgCUgUaC-CC[3′T] 188 0.3 32 0.7

[0240] TABLE 19 Truncates and 2′-OCH₃ purine modifications of nucleicacid ligand #13 from a biased SELEX. NAME SEQUENCE^(a) SEQ ID NO.BINDING^(b) LENGTH^(c) BIOACTIVITY^(d) NX22385UGUGAAUAUUAGAGAGUCUGUAUAGCUCUACCCC[3′T] 189 0.4 34 4 NX22386UgUgaAUaUUaGagagUCUGUAUagCUCUaCCCC[3′T] 190 3000 34 >100 NX22387UgUgaaUaUUagagagUCUgUAUagCUCUaCCCC[3′T] 191 3000 34 30 NX22424UgUgAAUAUUaGagagUCUGUAUAgCUCUaCCCC[3′T] 192 0.6 34 >100 NX22425UgUgaaUAUUagagagUCUGUAuAgCUCUaCCCC[3′T] 193 1.5 34 >100

[0241] TABLE 20 Pharmacokinetic properties of NX22323 in rats using anoncompartmental analysis. Parameter Units Estimate Cmax (μg/mL) 27.1AUClast ((μg * min)/mL) 3028.0 AUCINF ((μg * min)/mL) 3058.0 Beta t1/2(min) 630.9 Cl (mL/(min * kg)) 0.33 MRTINF (min) 350.4 Vss (mL/kg) 115.0Vz (mL/kg) 298.0

[0242] TABLE 21 Pharmacokinetic properties of NX22323 in rats using acompartmental analysis. Parameter Units Estimate StdError % Error Cmax(μg/mL) 16.3 3.3 20.2 AUCINF ((μg * min)/mL) 2486 274 11.0 Alpha-t1/2(min) 63.5 19.1 30.2 Beta-t1/2 (min) 467.2 83.2 17.8 A (μg/mL) 14.633.21 21.9 B (μg/mL) 1.70 0.84 49.1 Cl (mL/(min * kg) 0.402 0.044 11.0MRTINF (min) 360.3 35.6 9.9 Vss (mL/kg) 144.9 23.1 15.9

[0243] TABLE 22 Binding and inhibitory activity of 2′-Omethyl-andPegyl-modifications of lead TGFβ1 truncate ligand CD70 SEQ ID NO.Binding Bioactivity ChD70 GGGUGCCUUUUGCCUAGGUUGUGAUUUGUAACCUUCUGCCCA 216+++ +++ ChD70-m1 gggUgCCUUUUGCCUAGGUUGUGAUUUGUAACCUUCUGCCCA 194 +ChD70-m2 GGGUGCCUUUUgCCUaggUUGUGAUUUGUAACCUUCUGCCCA 195 ++ ChD70-m3GGGUGCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUGCCCA 196 +++ ChD70-m4GGGUGCCUUUUGCCUAGGUUGUGAUUUGUaaCCUUCUgCCCa 197 ++ ChD70-m5gGGUGCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUGCCCA 198 +++ ChD70-m6GgGUGCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUGCCCA 199 +++ ChD70-m7GGgUGCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUGCCCA 200 +++ ChD70-m8GGGUgCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUGCCCA 201 + ChD70-m9GGGUGCCUUUUgCCUAGGUUgUgaUUUgUAACCUUCUGCCCA 202 + ChD70-m10GGGUGCCUUUUGCCUaGGUUgUgaUUUgUAACCUUCUGCCCA 203 +++ ChD70-m11GGGUGCCUUUUGCCUAgGUUgUgaUUUgUAACCUUCUGCCCA 204 +++ ChD70-m12GGGUGCCUUUUGCCUAGgUUgUgaUUUgUAACCUUCUGCCCA 205 +++ ChD70-m13GGGUGCCUUUUGCCUAGGUUgUgaUUUgUaACCUUCUGCCCA 206 +++ ChD70-m14GGGUGCCUUUUGCCUAGGUUgUgaUUUgUAaCCUUCUGCCCA 207 +++ ChD70-m15GGGUGCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUgCCCA 208 +++ − ChD70-m16GGGUGCCUUUUGCCUAGGUUgUgaUUUgUAACCUUCUGCCCa 209 +++ ChD70-m17gggUGCCUUUUGCCUaggUUgUgaUUUgUaaCCUUCUGCCCa3′-3′U 210 +++ +++ ChD70-m18gggUGCCUUUUGCCUaggUUgUgaUUUgUaACCUUCUGCCCa3′-3′U 211 +++ ChD70-m19gggUGCCUUUUGCCUaggUUgUgaUUUgUaaCCUUCUGCCC3′-3′U 212 ++ − ChD70-m20gggUGCCUUUUGCCUaggUUgU-----gUaaCCUUCUGCCCa3′-3′U 213 ++ ChD70-m21gggUGCCUUUUGCCUaggUUg-------UaaCCUUCUGCCCa3′-3′U 214 ++ ChD70-m22gggUGCCUUUUGCCUaggUU---------aaCCUUCUGCCCa3′-3′U 215 +++

[0244]

1 216 1 71 DNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 1 tcgggcgagt cgtctgnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnccgc 60 atcgtcctcc c 71 2 32 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 2 taatacgactcactataggg aggacgatgc gg 32 3 16 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 3 tcgggcgagt cgtctg 16 4 88 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 4 taatacgact cactataggg aggacgatgc ggnnnnnnnn nnnnnnnnnnnnnnnnnnnn 60 nnnnnnnnnn nncagacgac tcgcccga 88 5 88 DNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 5attatgctga gtgatatccc tcctgctacg ccnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn nngtctgctg agcgggct 88 6 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 6 gggaggacgaugcggnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnncagac 60 gacucgcccg a71 7 65 DNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 7 gggaggacga tgcggnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnc agacgactcg 60 cccga 65 8 82 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 8 taatacgactcactataggg aggacgatgc ggnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnncagacgactcgccc ga 82 9 82 DNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 9 attatgctga gtgatatccc tcctgctacgccnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnngtct gctgagcggg ct 82 10 65RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 10 gggaggacga ugcggnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnncagacgacucg 60 cccga 65 11 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 11 cgcaggatcc taatacgact cactata27 12 38 DNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 12 ggcagaattc tcatctactt agtcgggcga gtcgtctg 38 13 23DNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 13 agcggataac aatttcacac agg 23 14 23 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 14 gtgctgcaaggcgattaagt tgg 23 15 19 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 15 actttatgct tccggctcg 19 16 19DNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 16 gccaaatgcc gagagaacg 19 17 18 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 17 ggggacaagcggacttag 18 18 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 18 gggagtacag ctatacag 18 19 15 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 19 ccgcaugcuc cuccc 15 20 14 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 20 ucgggcgagu cgcg14 21 61 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 21 gggaggacga ugcgguccuc aaugaucuuu ccuguuuaugcuccccagac gacucgcccg 60 a 61 22 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 22 gggaggacga ugcggaaguaacguuuaagu aaaauucguu cucucgguau uuggccagac 60 gacucgcccg a 71 23 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 23 gggaggacga ugcggaagua acguugaagu aaaauucguu cucucggcauuuggccagac 60 gacucgcccg a 71 24 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 24 gggaggacga ugcgguccuaaccaucacaa ucucaauucu uauauuuucc cgccccagac 60 gacucgcccg a 71 25 69 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 25 gggaggacga ugcggaaacc aaaagaccac aucuccauac ucacgcucugccccagacga 60 cucgcccga 69 26 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 26 gggaggacga ugcggauagaucgguccgau aagucuuuca ucuuuaccug gcccccagac 60 gacucgcccg a 71 27 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 27 gggaggacga ugcggaagua acguugaagu aaaauucguu cucucgguauuuggccagac 60 gacucgcccg a 71 28 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 28 gggaggacga ugcggacgauccuuuccuua acauuucauc auuucuccug ugccccagac 60 gacucgcccg g 71 29 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 29 gggaggacga ugcgguccau caacaaucuu aucauuaugu uuuuccuucccgccccagac 60 gacucgcccg a 71 30 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 30 gggaggacga ugcgguccucugagccgauc uucuucacua cuucuuuuuc ugccccagac 60 gacucgcccg a 71 31 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 31 gggaggacga ugcgguuccu caauucuucc aucuucauaa uguuucccuuugccccagac 60 gacucgcccg a 71 32 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 32 gggaggacga ugcggucuacccuuuagcag uauuuguuuc caucguuguu ugccccagac 60 gacucgcccg g 71 33 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 33 gggaggacga ugcggucuca acgaagaaca ucguuggaua cuguuugucccgccccagac 60 gacucgcccg a 71 34 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 34 gggaggacga ugcgguucaguuuccuucag uuuucguuuc uaauucuugu guccccagac 60 gacucgcccg a 71 35 61 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 35 gggaggacga ugcggagcgg auuaauuagu cugacuucuu guccccagacgacucgcccg 60 a 61 36 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 36 gggaggacga ugcggagacaucuuugucuc gauuagucau guuccuuacc ugccccagac 60 gacucgcccg a 71 37 69 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 37 gggaggacga ugcgguccuc uagcaagcag cuucucaucu uauuuuuccgccccagacga 60 cucgcccga 69 38 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 38 gggaggacga ugcggugcacagugauggau gacauuguau aacgguaugc guccccagac 60 gacucgcccg a 71 39 70 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 39 gggaggacga ugcggaccua ucuuucuucc aagucauagu uuuacuucccgccccagacg 60 acucgcccga 70 40 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 40 gggaggacga ugcggaugagaccuaaucau cgauccgcua ucuaaaaccu caccccagac 60 gacucgcccg a 71 41 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 41 gggaggacga ugcgguccuc agacaaaucu uucuugaauc uuuccuuaacugccccagac 60 gacucgcccg a 71 42 70 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 42 gggaggacga ugcggaccgauucuccaacu ugacauuuau uccucuuucu gccccagacg 60 acucgcccga 70 43 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 43 gggaggacga ugcgguccuc ugagccaauc uucuucgcua cuucuuuuucugccccagac 60 gacucgcccg a 71 44 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 44 gggaggacga ugcggauucuuucuccaacg cuuuucacua ccuacauuuc ugccccagac 60 gacucgcccg a 71 45 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 45 gggaggacga ugcggauccu auccucugaa uaucauuaaa ucaucuucuccgccccagac 60 gacucgcccg a 71 46 70 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 46 gggaggacga ugcgguucaaucaucuucac ucucauuucc uuuuuccuac uccccagacg 60 acucgcccga 70 47 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 47 gggaggacga ugcggcgaua gaaucuaguc guucuagaug aucugguacgugccccagac 60 gacucgcccg a 71 48 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 48 gggaggacga ugcgguaguaauccuugucu uccauuucuc uuuacccuuu ugccccagac 60 gacucgcccg a 71 49 61 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 49 gggaggacga ugcggcccau uaguccucau uaguccccug ugccccagacgacucgcccg 60 a 61 50 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 50 gggaggacga ugcggcaucuuauccuccau caguuacucu ucguuauucc cgccccagac 60 gacucgcccg a 71 51 70 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 51 gggaggacga ugcgguccaa auccucuucc cauguuagca uucagccuuguccccagacg 60 acucgcccga 70 52 70 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 52 gggaggacga ugcgguuccgacaauuuccu ccaccauuag auuucuugcu gccccagacg 60 acucgcccga 70 53 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 53 gggaggacga ugcggucuug auccuccuuu gugucuuucu uugucuucccugccccagac 60 gacucgcccg a 71 54 70 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 54 gggaggacga ugcggaaguaacguugaagu aaaauucguu cucucgguau uggccagacg 60 acucgcccga 70 55 70 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 55 gggaggacga ugcgguccga ucaguuccuu cgauuaaucu ucuuuccugccccccagacg 60 acucgcccga 70 56 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 56 gggaggacga ugcggaauccuucucccuga ugaauaugac cuuuuucuug cuccccagac 60 gacucgcccg a 71 57 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 57 gggaggacga ugcggaugau cuuuaauguc ugguuugagg ucaaugcgggugccccagac 60 gacucgcccg a 71 58 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 58 gggaggacga ugcggagaugguacuccauc uccuuuaugu gcccaucgcu guccccagac 60 gacucgcccg a 71 59 61 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 59 gggaggacga ugcgguccuc gauucuaauu uacuccuuuu ucccccagacgacucgcccg 60 a 61 60 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 60 gggaggacga ugcggucuacccuuuagcag uauuuguuuc caucguuguu ugccccagac 60 gacucgcccg a 71 61 70 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 61 gggaggacga ugcggcacaa uauucuccuc uacuuccacg uauuuuccuguccccagacg 60 acucgcccga 70 62 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 62 gggaggacga ugcgguccucaaccuuagac uuucauuucu ucaguucuuc ugccccagac 60 gacucgcccg a 71 63 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 63 gggaggacga ugcgguagug gucugucaaa ggaauagcua guaguguuugguccccagac 60 gacucgcccg a 71 64 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 64 gggaggacga ugcggcaucuuccuuagcau accaguuuau uccuuucccu guccccagac 60 gacucgcccg a 71 65 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 65 gggaggacga ugcggagcga caguauaguu aguacucuag cucuagugcuguccccagac 60 gacucgcccg a 71 66 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 66 gggaggacga ugcggaccucucaugaucag caucucgcgu aaucacgguu caccccagac 60 gacucgcccg a 71 67 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 67 gggaggacga ugcgguccgu acuccauuuc cuauuugauu ccuuuuccucugccccagac 60 gacucgcccg a 71 68 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 68 gggaggacga ugcggaacccacgaccuuac cuuaaucaug uauuucucuc ugccccagac 60 gacucgcccg a 71 69 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 69 gggaggacga ugcggagaua augagugacg gugauuauag augcugccccagacgacucg 60 cccga 65 70 71 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 70 gggaggacga ugcgguuccucaauucuucc aucuucauaa uguuucccuu ugccccagac 60 gacucgcccg a 71 71 60 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 71 gggaggacga ugcgguuccu uccaacguua ucuacuuucu gccccagacgacucgcccga 60 72 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 72 gggaggacga ugcggaagua acguuguaguaaaauucguu cucucggcau uuggccagac 60 gacucgcccg a 71 73 70 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 73gggaggacga ugcggaagua acguuauagu aaaauucguu cucucgguau uggccagacg 60acucgcccga 70 74 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 74 gggaggacgg ugcggaagua acguuguaguaaaauucguu cucucggcgu uuggccagac 60 gacucgcccg a 71 75 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 75gggaggacga ugcggaagua acguuguagu aaaauucguu cucucggcgu uuggucagac 60gacucgcccg a 71 76 70 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 76 gggagacgau gcggaaguaa cguuguaguaaaauucguuc ucucggcauu uggccagacg 60 acucgcccga 70 77 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 77gggaggacga ugcggaagua acguugaagu aaaauucguu cucucugcgu uuggucagac 60gacucgcccg a 71 78 70 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 78 gggaggacga ugcggaagua acguugaaguaaaauucguu cuccugguau uggccagacg 60 acucgcccga 70 79 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 79gggaggacga ugcggaagua acguugaagu aaaauucguu cucucggcau uuggccagac 60gacucgcccg a 71 80 70 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 80 gggaggacga ugcggaagua acguuguaguaaaauucguu cucuuggcau uugccagacg 60 acucgcccga 70 81 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 81gggaggacga ugcggaagua acguuaaagu aaaauucguu cucucggcgu uuggccagac 60gacucgcccg a 71 82 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 82 gggaggacga ugcggaagua acguugaaguaaaauucguu cucucggcgu uuggccagac 60 gacucgcccg a 71 83 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 83gggaggacga ugcggaagua acguuguagu aaaauucguu cucucgguau uuggccagac 60gacucgcccg a 71 84 70 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 84 gggaggacga ugcggaagua acguuguaguaaaauucguu cucuuggucu uggccagacg 60 acucgcccga 70 85 70 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 85ggaggacgau gcgaaguaac guuguaguaa aauucguucu cucgggcauu uggcagacga 60cuucgcccga 70 86 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 86 gggaggacga ugcggcguug uuuagucguauguauauacu aaguccgcuu guccccagac 60 gacucgcccg a 71 87 70 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 87gggaggacga ugcgguucag gagguuauua cagagucugu auagcuguac uccccagacg 60acucgcccga 70 88 70 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 88 gggaggacga ugcgguucag gaggguauuacagagucugu auagcuguac uccccagacg 60 acucgcccga 70 89 57 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 89gguucaggag guuauuacag agucuguaua gcuguacucc ccagacgacu cgcccga 57 90 56RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 90 gggaggacga ugcgguucag gagguuauua cagagucugu auagcuguacucccca 56 91 43 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 91 gguucaggag guuauuacag agucuguauagcuguacucc cca 43 92 36 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 92 ggagguuauu acagagucuguauagcugua cucccc 36 93 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 93 ggagguuauu acagagucuguauagcugua cucc 34 94 30 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 94 ggagguuauu acagagucuguauagcugua 30 95 26 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 95 ggagguuauu acagagucug uauagc 26 96 30 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 96 ggagguuauu acagagucug uauagccucc 30 97 30 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 97ggagguuauu agagucuaua gcuguacucc 30 98 26 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 98 ggagguuauuagagucuaua gccucc 26 99 33 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 99 ggagguuauu acagagucuguauagcugua cuc 33 100 32 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 100 ggagguuauu acagagucuguauagcugua cu 32 101 31 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 101 ggagguuauu acagagucuguauagcugua c 31 102 33 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 102 ggagguuauu acagagucug uauagcguac ucc 33103 33 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 103 ggagguuauu acagagucug uauagcuguc ucc 33 104 32RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 104 ggagguuauu acagagucug uauagcuacu cc 32 105 33 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 105 ggagguuaua cagagucugu auagcuguac ucc 33 106 33 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 106 ggagguuauu acagaucugu auagcuguac ucc 33 107 32 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 107 ggagguuauu acaaguugua uagcuguacu cc 32 108 33 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 108 ggagguuauu acagaguugu auagcuguac ucc 33 109 32 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 109 gggguuauua cagagucugu auagcuguac cc 32 110 32 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 110 ggagguuauu acgagucugu auagcguacu cc 32 111 33 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 111 ggagauauua cagagucugu auagcuguac ucc 33 112 30 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 112 gggguuauuc agagucugua uagcugaccc 30 113 30 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 113gggguuauua agagucugua uagcuuaccc 30 114 36 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 114 ggagguuauuacagagucug uauagcugua cucccc 36 115 34 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 115 ggagguuauuacagagucug uauagcugua cucc 34 116 37 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 116 ggagguuauu acagagucuguauagcugua cucccca 37 117 30 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 117 ggagguuauu acagagucuguauagcugua 30 118 33 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 118 gagguuauua cagagucugu auagcuguac ucc 33119 32 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 119 agguuauuac agagucugua uagcuguacu cc 32 120 31 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 120 gguuauuaca gagucuguau agcuguacuc c 31 121 34 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 121ggagguuauu acagagucug uauagcugua cucc 34 122 60 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 122 Ala Leu AspThr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys 1 5 10 15 Val ArgGln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 Ile HisGlu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 Pro TyrIle Trp Ser Leu Asp Thr Gln Tyr Ser Lys 50 55 60 123 60 PRT ArtificialSequence Description of Artificial Sequence Synthetic Sequence 123 AlaLeu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys 1 5 10 15Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp 20 25 30Ile His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys 35 40 45Pro Tyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg 50 55 60 124 60 PRTArtificial Sequence Description of Artificial Sequence SyntheticSequence 124 Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn CysCys 1 5 10 15 Val Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly TrpLys Trp 20 25 30 Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser GlyPro Cys 35 40 45 Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr 50 5560 125 52 PRT Artificial Sequence Description of Artificial SequenceSynthetic Sequence 125 Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly AlaSer Ala Ala Pro 1 5 10 15 Cys Cys Val Pro Gln Ala Leu Glu Pro Leu ProIle Val Tyr Tyr Val 20 25 30 Gly Arg Lys Pro Lys Val Glu Gln Leu Ser AsnMet Ile Val Arg Ser 35 40 45 Cys Lys Cys Ser 50 126 52 PRT ArtificialSequence Description of Artificial Sequence Synthetic Sequence 126 ValLeu Ser Leu Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro 1 5 10 15Cys Cys Val Ser Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile 20 25 30Gly Lys Thr Pro Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser 35 40 45Cys Lys Cys Ser 50 127 52 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 127 Val Leu Gly Leu Tyr Asn ThrLeu Asn Pro Glu Ala Ser Ala Ser Pro 1 5 10 15 Cys Cys Val Pro Gln AspLeu Glu Pro Leu Thr Ile Leu Tyr Tyr Val 20 25 30 Gly Arg Thr Pro Lys ValGlu Gln Leu Ser Asn Met Val Val Lys Ser 35 40 45 Cys Lys Cys Ser 50 12858 RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 128 ggaaguaacg uuguaguaaa auucguucuc ucggcauuug gccagacgacucgcccga 58 129 57 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 129 gggaggacga ugcggaagua acguuguaguaaaauucguu cucucggcau uuggcca 57 130 44 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 130 ggaaguaacguuguaguaaa auucguucuc ucggcauuug gcca 44 131 38 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 131 gggaggaugcggaaguaacg uuguaguaaa auuccuuc 38 132 35 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 132 gggaggaugcggaaguaacg uuguaguaaa auucc 35 133 32 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 133 gggaggaugcggaaguaacg uuguaguaaa au 32 134 33 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 134 gggaggaugc ggaaguaacguuguaguucc uuc 33 135 27 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 135 gggaggaugc ggaaguaacg uuguagu27 136 20 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 136 gggaggagua acguuguagu 20 137 58 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 137ggcguuguuu agucguaugu auauacuaag uccgcuuguc cccagacgac ucgcccga 58 13857 RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 138 gggaggacga ugcggcguug uuuagucgua uguauauacu aaguccgcuugucccca 57 139 44 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 139 ggcguuguuu agucguaugu auauacuaaguccgcuuguc ccca 44 140 44 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 140 ggggagcggc guuguuuagucguauguaua uacuaagucc gcuu 44 141 29 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 141 ggggagcggc guuguugaaaaguccgcuu 29 142 38 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 142 ggggagcggc guuguuucgu auguauauaaguccgcuu 38 143 33 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 143 ggggagcggc guuguuuaug uauaaguccg cuu 33144 42 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 144 ggggagcggc guuguuuagu cguauguaua uacuaagucc gc 42145 38 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 145 ggggagcggc guuguuuagu cguauguaua uacuaagu 38 14665 RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 146 gggaggacga ugcgggguga uuauuacaga guauguauag cuguacccccagacgacucg 60 cccga 65 147 64 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 147 gggaggacga ugcggaggcguuauuagaga gucuguauag cucuagcccc agacgacucg 60 ccga 64 148 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 148 gggaggacga ugcggggagg guauuacaga guauguauag cuguacucccagacgacucg 60 cccga 65 149 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 149 gggaggacga ugcggggagguuauuauaga gucuguauag cuauaccccc agacgacucg 60 cccga 65 150 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 150 gggaggacga ugcgggaggg uuauuauaga gucugcauag cuauacccccagacgacucg 60 cccga 65 151 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 151 gggaggacga ugcggugagaguauuacgga guauguauag ccguaccccc agacgacucg 60 cccga 65 152 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 152 gggaggacga ugcgggggca uuauuucaga gucuguauag cuguagccccagacgacucg 60 cccga 65 153 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 153 gggaggacga ugcgggcggauuaucacaga guauguauag cugugccgcc agacgacucg 60 cccga 65 154 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 154 gggaggacga ugcgguguga auauuagaga gucuguauag cucuacccccagacgacucg 60 cccga 65 155 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 155 gggaggacga ugcggcgggauuauuacuga gucuguauag caguaccccc agacgacucg 60 cccga 65 156 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 156 gggaggacga ugcgggugga auauuacgga gucuguauag ccguacucccagacgacucg 60 cccga 65 157 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 157 gggaggacga ugcggggggacuauuaguga gucuguauag cacuaccccc agacgacucg 60 cccga 65 158 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 158 gggaggacga ugcgggugga uuauuacagc gucuguauau cuguacccccagacgacucg 60 cccga 65 159 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 159 gggaggacga ugcgggcagguuauuacaga gucuguauag cuguacugcc agacgacucg 60 cccga 65 160 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 160 gggaggacga ugcgggguag auaucacuga gucuguauag caguguccccagacgacucg 60 cccga 65 161 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 161 gggaggacga ugcggagggauuauuacaga gucuguauag cuguaccccc agacgacucg 60 cccga 65 162 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 162 gggaggacga ugcgggugga uuauuacaga gucuguauag cuguacccccagacgacucg 60 cccga 65 163 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 163 gggaggacga ugcgggggcguuauuacaga gucuguauag cuguagcccc agacgacucg 60 cccga 65 164 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 164 gggaggacga ugcggggugg uuauuacaca guauguauag guguacccccagacgacucg 60 cccga 65 165 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 165 gggaggacga ugcggagggaauauuacaga guauguauag cuguaccccc agacgacucg 60 cccga 65 166 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 166 gggaggacga ugcggggagu uuauuacagc gucuguauau cuguagccccagacgacucg 60 cccga 65 167 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 167 gggaggacga ugcggugagguuauuacaga gucuguauag cuguacuccc agacgacucg 60 cccga 65 168 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 168 gggaggacga ugcggggugg uuauuagaga gucuguauag cucuacgcccagacgacucg 60 cccga 65 169 65 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 169 gggaggacga ugcggggggaguauuaaaga gucuguauag cuuuaccccc agacgacucg 60 cccga 65 170 65 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 170 gggaggacga ugcggggagg auauuauaga gucuguauag cuauacccccagacgacucg 60 cccga 65 171 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 171 ggagguuauu acagagucuguauagcugua cucc 34 172 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 172 ggagguuauu acagagucuguauagcugua cucc 34 173 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 173 ggagguuauu acagagucuguauagcugua cucc 34 174 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 174 ggagguuauu acagagucuguauagcugua cucc 34 175 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 175 ggagguuauu acagagucuguauagcugua cucc 34 176 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 176 ggagguuauu acagagucuguauagcugua cucc 34 177 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 177 ggagguuauu acagagucuguauagcugua cucc 34 178 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 178 ggagguuauu acagagucuguauagcugua cucc 34 179 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 179 ggagguuauu acagagucuguauagcugua cucc 34 180 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 180 ggagguuauu acagagucuguauagcugua cucc 34 181 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 181 ggagguuauu acagagucuguauagcugua cucc 34 182 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 182 ggagguuauu acagagucuguauagcugua cucc 34 183 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 183 ggagguuauu acagagucuguauagcugua cucc 34 184 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 184 ggagguuauu acagagucuguauagcugua cucc 34 185 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 185 ggagguuauu acagagucuguauagcugua cucc 34 186 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 186 ggagguuauu acagagucuguauagcugua cucc 34 187 33 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 187 gagauauua cagagucuguauagcuguac ucc 33 188 32 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 188 gggguuauua cagagucuguauagcuguac cc 32 189 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 189 ugugaauauu agagagucuguauagcucua cccc 34 190 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 190 ugugaauauu agagagucuguauagcucua cccc 34 191 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 191 ugugaauauu agagagucuguauagcucua cccc 34 192 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 192 ugugaauauu agagagucuguauagcucua cccc 34 193 34 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 193 ugugaauauu agagagucuguauagcucua cccc 34 194 42 RNA Artificial Sequence modified_base(1)..(42) A′s and g′s at positions 1-3 and 5 are 2′-OMe. 194 gggugccuuuugccuagguu gugauuugua accuucugcc ca 42 195 42 RNA Artificial Sequencemodified_base (1)..(42) A′s and g′s at positions 12 and 16-18 are 2′OMe.195 gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42 196 42 RNAArtificial Sequence modified_base (1)..(42) A′s and g′s at positions 21,23-24 and 28 are 2′-OMe. 196 gggugccuuu ugccuagguu gugauuugua accuucugccca 42 197 42 RNA Artificial Sequence modified_base (1)..(42) A′s and g′sat positions 30-31, 38 and 42 are 2′-OMe. 197 gggugccuuu ugccuagguugugauuugua accuucugcc ca 42 198 42 RNA Artificial Sequence modified_base(1)..(42) A′s and g′s at positions 1, 21, 23-24 and 28 are 2′-OMe. 198gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42 199 42 RNA ArtificialSequence modified_base (1)..(42) A′s and g′s at positions 2, 21, 23-24and 28 are 2′-OMe. 199 gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42200 42 RNA Artificial Sequence modified_base (1)..(42) A′s and g′s atpositions 3, 21, 23-24 and 28 are 2′-OMe. 200 gggugccuuu ugccuagguugugauuugua accuucugcc ca 42 201 42 RNA Artificial Sequence modified_base(1)..(42) A′s and g′s at positions 5, 21, 23-24 and 28 are 2′-OMe. 201gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42 202 42 RNA ArtificialSequence modified_base (1)..(42) A′s and g′s at positions 12, 21, 23-24and 28 are 2′-OMe. 202 gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42203 42 RNA Artificial Sequence modified_base (1)..(42) A′s and g′s atpositions 16, 21, 23-24 and 28 are 2′-OMe. 203 gggugccuuu ugccuagguugugauuugua accuucugcc ca 42 204 42 RNA Artificial Sequence modified_base(1)..(42) A′s and g′s at positions 17, 21, 23-24 and 28 are 2′-OMe. 204gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42 205 42 RNA ArtificialSequence modified_base (1)..(42) A′s and g′s at positions 18, 21, 23-24,and 28 are 2′-OMe. 205 gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42206 42 RNA Artificial Sequence modified_base (1)..(42) A′s and g′s atpositions 21, 23-24, 28 and 30 are 2′-OMe. 206 gggugccuuu ugccuagguugugauuugua accuucugcc ca 42 207 42 RNA Artificial Sequence modified_base(1)..(42) A′s and g′s at positions 21, 23-24, 28 and 31 are 2′-OMe. 207gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42 208 42 RNA ArtificialSequence modified_base (1)..(42) A′s and g′s at positions 21, 23-24, 28and 38 are 2′-OMe. 208 gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42209 42 RNA Artificial Sequence modified_base (1)..(42) A′s and g′s atpositions 21, 23-24, 28 and 42 are 2′-OMe. 209 gggugccuuu ugccuagguugugauuugua accuucugcc ca 42 210 42 RNA Artificial Sequence modified_base(1)..(42) A′s and g′s at positions 1-3, 16-18, 21, 23-24, 28, 30-31 and42 are 2′-OMe; linkage at positions 42 and 43 is 3′-3′. 210 gggugccuuuugccuagguu gugauuugua accuucugcc ca 42 211 42 RNA Artificial Sequencemodified_base (1)..(42) A′s and g′s at positions 1-3, 16-18, 21, 23-24,28, 30 and 42 are 2′-OMe; linkage at positions 42 and 43 is 3′-3′. 211gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42 212 41 RNA ArtificialSequence modified_base (1)..(41) A′s and g′s at positions 1-3, 16-18,21, 23-24, 28 and 30-31 are 2′-OMe; linkage at positions 41 and 42 are3′-3′. 212 gggugccuuu ugccuagguu gugauuugua accuucugcc c 41 213 37 RNAArtificial Sequence modified_base (1)..(37) A′s and g′s at positions1-3, 16-18, 21, 23, 25-26 and 37 are 2′-OMe; linkage at positions 37 and38 is 3′-3′. 213 gggugccuuu ugccuagguu guguaaccuu cugccca 37 214 35 RNAArtificial Sequence modified_base (1)..(35) A′s and g′s at positions1-3, 16-18, 21, 23-24 and 35 are 2′-OMe; linkage at positions 35 and 36is 3′-3′. 214 gggugccuuu ugccuagguu guaaccuucu gccca 35 215 33 RNAArtificial Sequence modified_base (1)..(33) A′s and g′s at positions1-3, 16-18, 21-22, and 33 are 2′-OMe; linkage at positions 33 and 34 is3′-3′. 215 gggugccuuu ugccuagguu aaccuucugc cca 33 216 42 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 216gggugccuuu ugccuagguu gugauuugua accuucugcc ca 42

We claim:
 1. A purified and isolated non-naturally occurring RNA ligandto TGFβ2 wherein said ligand is selected from the group consisting ofthe sequences set forth in Tables 5, 7, 8, 11, 13, 14, 16-19, and FIG. 9(SEQ ID NOS: ).
 2. A Complex comprised of a TGFβ2 Nucleic Acid Ligandand a Non-Immunogenic, High Molecular Weight Compound or LipophilicCompound.
 3. The Complex of claim 2 further comprising a Linker betweensaid Ligand and said Non-Immunogenic, High Molecular Weight Compound orLipophilic Compound.
 4. The Complex of claim 2 wherein saidNon-Immunogenic, High Molecular Weight Compound is a PolyalkyleneGlycol.
 5. The Complex of claim 4 wherein said Polyalkylene Glycol ispolyethylene glycol (peg).
 6. The Complex of claim 5 wherein said PEGhas a molecular weight of about between 10-80 K.
 7. The Complex of claim6 wherein said PEG has a molecular weight of about 20-45 K.
 8. TheComplex of claim 7 wherein said Complex is

LIGAND=rGrGrArGrGfUfUrAfUfUrAfCrArGrArGfUfCfUrGfUfUrArGfCfUrGfUrAfCfUfCfC-3′-3′-dT